2. a longer half-life of peptide-HSP70 interaction; in fact, data sug-
gest that one of the putative receptors for HSP70, CD40, will only
bind HSP70 strongly in the presence of the peptide substrate (31),
which is facilitated by the higher affinity interaction between the
peptide and HSP.
Evaluation of the binding kinetics of the original Javelin-hybrid
epitope constructs revealed that while J0 substantially enhanced
the affinity between the hybrid peptide and HSP70, there was still
a high degree of variability in the binding affinities of Javelin-
hybrid peptide constructs containing various antigenic epitopes. As
a result, we sought to improve the hybrid peptide design to make
the Javelin a more universal affinity-enhancing sequence. In this
study, we report that by creating hybrid epitopes with a higher
HSP70-binding affinity Javelin (J1), modifying the linker sequence
to enhance Ag processing, and moving the Javelin-linker sequence
to the N-terminal side of the epitope, we were able to dramatically
improve the magnitude of immune responses to several mouse and
human antigenic epitopes in the absence of conventional adjuvant.
Moreover, the uniform, high-affinity interaction allows for equal
delivery of multiple epitopes in a single formulation with the po-
tential for highly successful therapeutic and prophylactic vaccines
for treatment and prevention of infectious diseases and cancer.
Materials and Methods
Mice and cell lines
Six- to 12-wk-old female C57BL/6 mice were obtained from the National
Cancer Institute Animal Resource Center (Frederick, MD) or The Jackson
Laboratory. TAPϪ/Ϫ
mice were obtained from The Jackson Laboratory.
HHDII mice (35) were obtained from F. Lemonnier at the Institute Pasteur
and bred at Charles River Laboratories. Mice were housed at New York
Medical College or at Antigenics, and cared for following the guidelines of
the Institutional Animal Care and Use Committee. The mouse thymoma
cell line EL4 and its E.G7 derivative (EL-4 transfected with cDNA encod-
ing OVA) were obtained from American Type Culture Collection and cul-
tured according to the supplier recommendations. The B3Z T-T hybridoma
(specific for the peptide SIINFEKL presented in the context of H2-Kb
) was
previously described (34).
Peptides and proteins
Peptides were purchased from New England Peptide or CS Bio and were
Ͼ96% pure. Table I shows the panel of peptides that constructed for these
experiments.
BSA fraction V (BSA) was purchased from Invitrogen Life Technolo-
gies. Phosphorylase B was purchased from Sigma-Aldrich and reconsti-
tuted in 50 mM HEPES with 150 mM NaCl, then filtered through a 45-m
filter. Recombinant human cytosolic HSP70 was prepared as described
previously (34).
Binding assay for hybrid peptide and HSP70
Eighty-microliter binding reactions were set up that spanned a dilution
series of the competitor peptide of interest and controls. Binding reactions
contained fluoresceinated peptide at a constant concentration, a varying
range of unlabeled competitor peptide, and a constant amount of HSP70 in
PBS plus 1 mM ADP (Crescent Chemical). The fluorescent peptide used in
the binding assay was fluorescently labeled P2-L1-J0: ALFDIESKVGS
GHWDFAWPW. Fluorescein was covalently attached to the N terminus,
strategically located on the Ag sequence and away from the HSP70-bind-
ing sequence. Reactions were incubated for 1 h at 25°C. A total of 25 l
of each reaction mix was then centrifuged through Microspin G-50 col-
umns (Amersham Biosciences). The column is designed to retain free pep-
tide while allowing protein and presumed protein-peptide complexes to
pass through. For every peptide tested, a control containing peptide in the
absence of HSP70 was used to ensure that all peptides were quantitatively
retained in the G-50 column. Upon centrifugation, material passing through
the columns was examined by fluorometry and by Bradford assay and no
fluorescence of or presence of protein was observed when HSP70 was not
Table I. Peptides used in this study
Ag Peptide Amino Acid Sequence MHC
OVA OVA SIINFEKL H2-Kb
OVA-L1-J0 SIINFEKLgsgHWDFAWPW
OVA-L1-J1 SIINFEKLgsgNLLRLTGW
OVA-J1 SIINFEKLNLLRLTGW
J1-L1-OVA NLLRLTGWgsgSIINFEKL
J1-L2-OVA NLLRLTGWffrkSIINFEKL
L2-OVA ffrkSIINFEKL
Bovine ␣-casein Bcas IAYFYPEL H2-Kb
J1-L2-Bcas NLLRLTGWffrkIAYFYPEL
Sendai virus SdV FAPGNYPAL H2-Kb
J1-L2-SdV NLLRLTGWffrkFAPGNYPAL
Vesicular stomatitis virus VSV RGYVYQGL H2-Kb
J1-L2-VSV NLLRLTGWffrkRGYVYQGL
Melanoma gp100 IMD IMDQVPFFS HLA 2.1
J1-L2-IMD NLLRLTGWffrkIMDQVPFFS
IMD-L1-J0 IMDQVPFFSgsgHWDFAWPW
IMD-L1-J1 IMDQVPFFSffrkNLLRLTGW
Melanoma tyrosinase YMD YMDGTMSQV HLA 2.1
J1-L2-YMD NLLRLTGWffrkYMDGTMSQV
YMD-L1-J0 YMDGTMSQVgsgHWDFAWPW
YMD-L1-J1 YMDGTMSQVgsgNLLRLTGW
Melanoma MART-1 MelA AAGIGILTV HLA 2.1
J1-L2-MelA NLLRLTGWffrkAAGIGILTV
Melanoma Trp-2 Trp2 SVDYFFVWL HLA 2.1
J1-L2-Trp2 NLLRLTGWffrkSVYDFFVWL
Prostate PSMA-P2 P2 ALFDIESKV HLA 2.1
P2-L1-J0 ALFDIESKVgsgHWDFAWPW
P2-L1-J1 ALFDIESKVgsgNLLRLTGW
CMV CMV NLVPMVATV HLA 2.1
CMV-L1-J0 NLVPMVATVgsgHWDFAWPW
CMV-L1-J1 NLVPMVATVgsgNLLRLTGW
Epstein Barr virus EBV GLCTLVAML HLA 2.1
EBV-L1-J0 GLCTLVAMLgsgHWDFAWPW
EBV-L1-J1 GLCTLVAMLgsgNLLRLTGW
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3. present in the binding solution, confirming that free peptides were fully
retained on the column.
Material passing through the columns was diluted in 8 M urea and
incubated at 95°C for 5 min to ensure dissociation of complexes. Samples
were then cooled on ice and analyzed by Bradford assay and fluorometry.
Fluorescence in 8 M urea was different from in buffer alone, so to correct
for this, a standard curve of P2-L1-J0 was made in 8 M urea and the
fluorescence from the binding reactions was measured against this standard
curve. Data were analyzed by determining the Hill coefficient: the IC50 of
the competitor ligand was determined by graphing log [Y/(1 Ϫ Y)] on the
y-axis vs log [S] on the x-axis, where Y is the known ligand bound and S
is the concentration of the competitor ligand. The IC50 was then used to
extrapolate the Ki of the competitor ligand: Ki ϭ IC50/[1 ϩ (Y/Kd)].
The dissociation constant of P2-L1-J0 with HSP70 was determined by
using a similar binding assay to that described for the competitor binding
assay, except a range of P2-L1-J0 concentrations were incubated with ex-
cess HSP70 in the absence of competitor peptide. The Kd was determined
by plotting the fraction of bound HSP70 over the concentration of free
P2-L1-J0 vs the fraction of bound HSP70. The Kd is the negative inverse
of the slope of the resulting line.
HSP70/Javelin-hybrid peptide complex preparation
For preparation of complexes of HSP70 coupled with hybrid peptides, the
indicated amounts of HSP70 and peptide were mixed in PBS (pH 7.4;
Zymed Laboratories) and incubated for 1 h at 25°C. After the 1-h incuba-
tion, 0.1 mM yeast-derived ADP (Crescent Chemical) was added, and the
complexes incubated at 25°C for an additional 30 min. All control samples,
including nonchaperone protein samples, were treated in the same manner
as the complex samples. After the final incubation, complexes were im-
mediately transferred to ice until used.
In vitro cross-presentation assay
Mice were injected i.p. with 1 cc 3% brewer thioglycolate yeast (Sigma-
Aldrich). Five days later, mice were euthanized and peritoneal exudate
cells were harvested by peritoneal lavage with ice-cold PBS (Invitrogen
Life Technologies). Cells were plated at 2 ϫ 106
/ml in flat-bottom 96-well
plates in serum-free medium (AIM-V; Invitrogen Life Technologies).
Plates were incubated at 37°C, 5% CO2 for 1 h, and then nonadherent cells
were washed off with 37°C medium. Complexes or control samples were
added to triplicate wells, and then the B3Z T-T hybridoma was added at a
final concentration of 5 ϫ 105
cells/ml in 200 l of AIM-V. Plates were
incubated overnight at 37°C in 5% CO2, then cell-free supernatants were
harvested. Supernatants were stored at Ϫ80°C until analyzed for IL-2 lev-
els using the Opt-EIA IL-2 ELISA kit (BD Pharmingen), according to
manufacturer’s instructions with the exception that all indicated volumes
were halved.
Immunization, ELISPOT, and 51
Cr-release assays
For mouse immunization, 50 l of complex or control solutions was in-
jected s.c. into the base of the tail. Seven days later, spleens were harvested
and either put directly into the ex vivo ELISPOT assay (details below) or
restimulated at 1–2 ϫ 107
responder cells/flask in the presence of 1–2 ϫ
107
peptide-pulsed, irradiated (3000 rad) normal syngeneic spleen cells in
RPMI 1640 containing 10% FCS (cRPMI-10%; HyClone), supplemented
with 2 mM L-glutamine, 100 m of penicillin-100 g of streptomycin, 0.1
mM MEM nonessential amino acids, 1 mM sodium pyruvate, and 50 M
2-ME (Invitrogen Life Technologies). Peptide pulsing was performed by
incubation of spleen cells with 10 g/ml peptide for 30 min at room tem-
perature. After 5 days of incubation, cytotoxic activity of restimulated cells
was measured in a standard 4-h 51
Cr-release assay. EL4 target cells were
labeled with 100 Ci of sodium [51
Cr]chromate and incubated with or
without 1 g/ml peptide for 1 h then extensively washed and used as target
cells. Specific lysis was determined using the following formula: percent-
specific release ϭ 100 ϫ (release by effector cells Ϫ spontaneous release)/
(maximal release Ϫ spontaneous release). Spontaneous release was Ͻ30%
maximum release in all experiments.
For the IFN-␥ ELISPOT assay, polyvinylidene difluoride membrane
microtiter plates (Millipore) were coated with 10 g/ml capture Ab
(Mabtech) and incubated overnight at 4°C. The following day, wells were
washed and blocked with cRPMI-10%. Spleens were pooled within groups
and CD8ϩ
T cells were enriched via the MidiMACS cell separation system
(Miltenyi Biotec) following the manufacturer’s instructions. Briefly, RBC
were lysed with ACK buffer, and the remaining cells were incubated with
anti-CD8 microbeads for 20 min at 4°C. Cells were washed once then
applied over the MidiMACS column attached to a magnet. Columns were
washed four times, and the flow-through was discarded. The CD8ϩ
T cells
were plunged off the column with 6 ml of buffer, washed, and resuspended
in complete medium. The enrichment success was routinely between 92
and 98% (data not shown). Between 2.5 and 4 ϫ 105
cells were plated per
well of the ELISPOT plate, as indicated in the results. Relevant or irrele-
vant peptide was added for a final concentration of 10 g/ml (negative and
positive control wells contained medium or 5 g/ml Con A, respectively).
In some experiments, peptide-pulsed naive splenocytes were used as APC
at a concentration of 5 ϫ 105
cells/well. Plates were wrapped in foil and
incubated for 18 h in a 37°C, humidified chamber with 5% CO2. For spot
development, wells were extensively washed, then incubated with biotin-
ylated detection Ab (Mabtech) for 2 h at 37°C. The wells were then washed
and Vectastain ABC peroxidase (Vector Laboratories) was added and
plates incubated at room temperature °C for 1 h. Spots were visualized after
incubation with AEC (Sigma-Aldrich) for 4 min, washed with tap water,
then dried. Analysis was performed on the CTL Immunospot Reader (Cel-
lular Technology) or by Zellnet Consulting.
Tumor challenge experiments
A total of 7.5 ϫ 105
E.G7 cells was injected s.c. into the right flank of mice
that had been immunized 14 and 7 days previously s.c. at the base of the
tail. Tumor growth was monitored every 3–4 days, and the results were
expressed as the mean volume (mm3
) calculated from the longest and its
perpendicular diameter of the tumor.
Results
The Javelin sequence confers the ability of antigenic epitopes to
form a stable complex with HSP70
We propose that the immune response to peptides with high af-
finity for HSP70 will be greater in magnitude than the response to
low-affinity peptides because higher affinity peptides are more
likely to remain in complex with HSP70 long enough to be chap-
eroned into APCs. Thus, any epitope modified to contain a Javelin
sequence should bind HSP70 with uniformly high affinity and be
more immunogenic when injected in vivo compared with the un-
modified epitope. To examine this likelihood, several hybrid pep-
tide constructs containing antigenic epitopes colinearly synthe-
sized with a flexible linker (GSG) and the Javelin J0 sequence
(HWDFAWPW; Ref. 33) were created. These peptide constructs
were first analyzed for their HSP70-binding kinetics in the pres-
ence of ADP, as described in Materials and Methods. All Javelin-
hybrid peptides displayed higher affinities for HSP70 than their
unmodified epitope counterpart; however, the relative affinities be-
tween different hybrid peptides were quite variable (Table II, “No
Javelin” vs “ϩ J0”). Moreover, while the specific immunogenicity
of the model hybrid Ag OVA-L1-J0 complexed with HSP70 was
consistently greater than complexes of HSP70 and the minimal
epitope OVA (OVA-L1-J0 and OVA tested at molar equivalents),
studies demonstrated considerable variability in the magnitude of
the responses to the hybrid Ag (data not shown).
The variability in binding and immunogenicity that was char-
acteristic of the hybrid peptides containing J0 led to the hypothesis
that even though these peptides individually bind HSP with higher
affinity than the unmodified epitopes, they may still not bind with
Table II. Dissociation constants of peptide constructs
Peptide Abbreviation
No Javelin
(M)
ϩ J0
(M)a
ϩ J1
(M)a
L1-J0 120
J0 118
J1 0.9
OVA OVA 236 26 0.8
Melanoma Tyrosinase YMD 209 20 1.9
Melanoma gp100 IMD 2567 180 0.5
Prostate PSMA-P2 P2 108 23 0.9
CMV CMV 132 7.5 3.6
Epstein Barr virus EBV 127 4.2 1.6
a
The epitopes were joined with the Javelin sequence by the L1 linker -GSG-.
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4. a high enough affinity for uniformity of responses in vivo, and they
may not all induce robust immune responses when immunized in
an HSP-based polyvalent vaccine. The unevenness in the binding
kinetics could result in one epitope being delivered with higher
frequency to APCs than another, due to the longer interaction with
HSP70. As a result, a new Javelin sequence was created that binds
HSP70 with a higher affinity than the J0 sequence, and when syn-
thesized in a string with antigenic epitopes, was predicted to stan-
dardize the affinity between the epitopes and HSP70. When the
new Javelin construct, J1 (NLLRLTGW; Refs. 36–40), was ana-
lyzed in the kinetic assay, the data revealed a 131-fold greater
affinity for HSP70 than J0 (Table II). To determine whether the
new Javelin facilitated uniformly higher affinity interactions be-
tween HSP70 and epitope, hybrid peptides were synthesized that
contained defined epitopes in sequence with the flexible linker
-GSG- (L1) and J1, then tested in the binding assay. A Hill plot
showing representative data from the mouse MHC class I Kb
bind-
ing epitope, SINFEKL, from hen egg OVA, is shown in Fig. 1. The
dissociation constant for OVA-L1-J0 was extrapolated to be 26
M whereas the Kd for the OVA-L1-J1 construct was 0.8 M, a
32-fold difference in affinity. Both affinities were substantially
greater than the 236 M dissociation constant for OVA. The ad-
ditional results for several human HLA-A*0201-binding epitopes
are also shown in Table II. The increase in the affinities of each of
these hybrid-J1 peptides over their unmodified counterpart ranged
from 37-fold for the CMV epitope to 5100-fold for the gp100
epitope. In fact, all of the sequences containing J1 exhibited low
micromolar/high nanomolar dissociation constants for HSP70 sig-
nifying a substantial improvement in binding affinity. These data
indicate that both murine and human epitopes can be modified with
the -L1-J1 sequence and bind HSP70 with higher affinity than the
minimal epitope. Synthesizing epitopes with the addition of the J1
sequence creates Javelin-hybrid peptide constructs that more uni-
formly bind HSP70 and as a result possibly provide stronger prim-
ing of immune responses, especially for the epitopes that are nat-
urally poor HSP70 binders like gp100 (Tables I and II).
High-affinity HSP70:Javelin-hybrid peptide complexes are better
at cross-presentation and induce stronger immune responses
than unmodified peptide
The OVA-Javelin hybrid peptides containing the J0 or J1 sequence
were tested in an in vitro cross-presentation assay to compare their
processing and presentation by APCs. As shown in Fig. 2A, at the
doses used, each of the Javelin-hybrid peptides alone, without ex-
ogenously added HSP70 can be processed and presented by APCs
at very low levels; however, the amount of IL-2 detected was not
significantly different from the nonspecific stimulation of the B3Z
T-T hybridoma by APCs treated with HSP70 without peptide.
When delivered in complex with HSP70, both Javelin-hybrid pep-
tide constructs could be processed, and the antigenic epitope pre-
sented by activated murine macrophages. On average, APCs
pulsed with 40 nM (85 ng/ml) OVA-L1-J1 in complex with 400
nM (28 g/ml) HSP70 induced approximately twice as much IL-2
from the B3Z cell line than the molar equivalent of OVA-L1-J0 in
complex with HSP70 ( p ϭ 0.024; Fig. 2A). Analysis of levels of
intracellular -galactosidase showed the same pattern as IL-2 pro-
duction (data not shown). These data indicate that the higher af-
finity J1 sequence improves the cross-presentation of Javelin-hy-
brid peptide, presumably by shifting the equilibrium of the
FIGURE 1. Hill plot for calculation of dissociation constants of OVA,
OVA-L1-J0, and OVA-L1-J1. Various concentrations of SIINFKEL
(OVA), OVA-L1-J0, or OVA-L1-J1 hybrid peptides were titrated into
binding reactions containing constant amounts of both HSP70 and a la-
beled reporter peptide of known affinity for HSP70. The abilities of these
peptides to compete out the binding of the reporter were analyzed using a
Hill plot and the IC50 of each determined as the point where the plot
intersected the y-axis. The Kd of each peptide was then calculated from its
experimentally determined IC50. [S], Concentration of hybrid peptide; Y,
fraction of labeled reporter peptide bound.
FIGURE 2. Cross-presentation of the OVA epitope delivered as a hy-
brid peptide complex with HSP70. A, Adherent PEC from thioglycolate-
induced mice were pulsed with 40 nM peptides, alone or in complex with
400 nM HSP70, then cocultured with the B3Z T-T hybridoma as described
in Materials and Methods. Pooled data from three experiments are shown,
represented as mean supernatant IL-2 quantities Ϯ SE. The p value was
calculated using the Student’s t test. B, Mice were immunized with 17.5
M (2 g) OVA-L1-J0 in complex with 3.6 M (12.5 g) HSP70 or 3.8
M (0.4 g) OVA-L1-J1 in complex with 0.74 M (2.6 g) HSP70 or
controls in a 50-l volume; 7 days later splenic CD8 T cell responses were
evaluated by ELISPOT with peptide pulsed naive splenocytes as APC.
Representative data (mean IFN-␥ spot number Ϯ SD) for greater than four
experiments is shown.
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5. interaction so that more Javelin-hybrid peptide is bound to HSP70
in a given formulation.
Next, the immunization capacity of the HSP70-Javelin hybrid
peptide vaccine was evaluated in C57BL/6 mice. It was hypothe-
sized that the greater cross-presentation that occurred with HSP70:
OVA-L1-J1 complex compared with HSP70:OVA-L1-J0 complex
would enable use of a lower dose of the former complex to achieve
the same response as a higher dose of the latter. The difference in
dosing requirements could be a direct result of the higher affinity
interaction between the J1-containing hybrid peptide and HSP70,
and hence a longer interaction time with more peptide bound at
equilibrium. Indeed, even when mice were immunized with HSP70:
OVA-L1-J1 at a 5-fold lower dose than HSP70:OVA-L1-J0, equiv-
alent results were obtained in the ex vivo IFN-␥ ELISPOT (Fig. 2B).
Mice immunized with the equivalent low dose of the HSP70:OVA-
L1-J0 did not have a measurable response in the assay (data not
shown). Additionally, the immunological activity of complexes of
HSP70:OVA-L1-J1 tested over many experiments was much less
variable than the J0 modified counterpart. Taken together, these data
indicate that the higher affinity J1-hybrid peptide in complex with
HSP70 consistently induces more vigorous immune responses and
better cross-presentation than the previously used, lower affinity J0-
hybrid peptide:HSP70 complex.
A novel cleavable linker sequence ensures optimal Ag
processing leading to the amplification of immune responses
The epitope contained within the J1-hybrid peptide constructs
needs to be processed by the intracellular machinery of APCs to be
presented by MHC class I molecules on the cell surface. The orig-
inal Javelin constructs contained a flexible -GSG- linker, but it
became apparent that further improvements in immunogenicity
might be achieved by creating a cleavable linker sequence between
the epitope and J1 that will be accessible to intracellular enzymes.
The linker sequence FFRK (L2) was designed to contain both ca-
thepsin and proteasomal cleavage sites (41–45). In addition, a pep-
tide was created in which the J1-linker sequence was reoriented to
the N-terminal side of the epitope and linker, based on the pub-
lished data that peptides with a C-terminal Javelin (BiP) appear to
be processed via the proteasomal-processing pathway, but peptides
with an N-terminal Javelin are processed via an endosomal-pro-
cessing pathway (21). Controls, or the new L2 sequence-contain-
ing constructs, in complex with HSP70 were used to immunize
mice, and the results compared with responses from mice immu-
nized with complexes formed with the other hybrid peptides (pep-
tide sequences shown in Table I). T cell responses from immu-
nized animals were analyzed either by ex vivo IFN-␥ ELISPOT
(Fig. 3A), or by a standard 51
Cr-release assay as described in Ma-
terials and Methods (Fig. 3B). The ex vivo IFN-␥ ELISPOT assay
revealed that the most remarkable immune response was detected
in mice immunized with HSP70:J1-L2-OVA complexes. The num-
bers of IFN-␥-secreting CD8ϩ
T cells were increased by Ͼ5-fold
compared with the number induced by the OVA epitope immu-
nized in adjuvant (TiterMax, Fig. 3A). The other Javelin-hybrid
peptide constructs complexed to HSP70 gave responses compara-
ble to the OVA plus TiterMax immunization. This result shows
that by including a cleavable linker and changing the orientation of
the Javelin-hybrid peptide construct, the responder frequency in-
creases to ϳ50 per 100,000 CD8ϩ
T cells. It is important to note
that in the ex vivo ELISPOT assay, HSP70 complexes containing
the OVA-J1 sequence without the L1 linker exhibited the same
level of response as the OVA-L1-J1 construct (Fig. 3A), thus con-
firming that L1 was not a readily cleavable linker and that it did not
have a large influence on the hybrid peptide processing. The fact
that the minimal OVA epitope in complex with HSP70 (HSP:
OVA) elicits a T cell response of similar magnitude to the OVA-J1
and OVA-L1-J1 peptides complexed to HSP70 is likely attribut-
able to cell surface loading of the epitope onto MHC class I mol-
ecules in the case of the former immunogen and the suboptimal
attributes of the extended peptides in the case of the latter immu-
nogens. The most relevant comparisons to assess in this experi-
ment are among the J1-L2-OVA peptide and the OVA-L1-J1 or
J1-L1-OVA peptides where in all cases MHC class I surface load-
ing is unlikely and where the enhanced immunogenicity associated
with the optimized Javelin (J1), linker (L2), and orientation (N
terminus) is clear.
The corresponding CTL assay showed the highest cytotoxic ac-
tivity was induced in mice immunized with HSP70:J1-L2-OVA
complex, followed by HSP70:OVA-L1-J1 complex (Fig. 3B). The
differences in rank in potency between the constructs as evaluated
by the ex vivo ELISPOT and the 51
Cr-release assay may reflect the
change in frequency of reactive CD8 T cells that occur as a result
FIGURE 3. Evaluation of new linker/orientation epitopes in vivo.
C57BL/6 mice were immunized s.c. at the base of the tail with 19 M (ϳ2
g) hybrid peptide complexed with 1.3 M (4.4 g) HSP70 or the appro-
priate controls in 50 l of saline containing 0.1 mM ADP, as described in
Materials and Methods. Seven days later, mice were euthanized and the
spleens harvested for analysis. A, CD8ϩ
T cells were enriched from one-
half a spleen of each immunized mouse and put into an ex vivo ELISPOT
assay to measure epitope-specific IFN-␥ production. Inset, The Ags used to
pulse naive splenic APC during in vitro stimulation and data are shown as
the mean IFN-␥ spot number Ϯ SD for three mice per group. B, Epitope-
specific CTL responses to complexes formed with new Javelin-hybrid
epitope constructs after one restimulation in vitro from the same mice
shown in A. Inset, The constructs used for immunization. Cytotoxicity
against SIINFEKL-pulsed EL4 cells is plotted, and killing of irrelevant
peptide pulsed targets did not exceed 10%. Data are average of three mice
per group from one representative experiment.
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6. of the in vitro expansion of CTL for the 51
Cr-release assay. Re-
gardless of the differences between the two assays, J1-L2-OVA
was the most effective construct tested in both assays. Among the
least potent immunogens in these experiments was HSP70:OVA,
which is also intriguing because the epitope does not need to be
processed before loading onto MHC class I molecules. Most
likely, the low affinity binding between OVA and HSP70 resulted
in fewer OVA peptides complexed with HSP70 at the given dose,
which was insufficient when compared with the molar equivalent
of the Javelin-hybrid peptides. Taken together, these data indicate
that designing the cleavable linker to facilitate the processing of
Javelin-hybrid peptide constructs and changing the orientation of
the hybrid constructs created a more efficient HSP70 peptide com-
plex vaccine that is able to induce vigorous immune responses
in vivo.
J1-L2-hybrid peptide processing can occur independently of the
proteasome
There has been much attention in the recent literature on the means
by which exogenous Ags are delivered into the endogenous path-
way of Ag processing and loaded onto MHC class I molecules.
Evidence exists for endogenous Ags delivered to MHC class I
molecules by both the proteasomal pathway of Ag processing,
feeding peptides through the TAP transporter into the endoplasmic
reticulum for loading onto MHC class molecules, and by the en-
dosomal route, where MHC class I molecules are recycled through
the endosomes to the cell surface (41). To determine whether the
processing of the new J1-L2-OVA peptide required the protea-
some or was dependent on the TAP transporter for loading onto
MHC class I molecules, cross-presentation in the presence of the
proteasome inhibitor lactacystin or by peritoneal exudate cells
(PECs) from TAPϪ/Ϫ
mice was evaluated. As shown in Fig. 4A,
the HSP70:OVA-L1-J1 complex required the proteasome for pro-
cessing, because cross-presentation was significantly inhibited in
the presence of 20 M lactacystin, verifying published observa-
tions with HSP70:OVA-L1-J0 (21). In contrast, there was no dif-
ference in the cross-presentation of HSP70:J1-L2-OVA between
the untreated and lactacystin-treated samples, indicating that there
was no requirement for passage through the proteasome. This re-
sult is not surprising, because the proteasome is required for proper
C-terminal cleavage of epitopes, and the J1-L2-OVA peptide al-
ready has the correct C terminus. In addition, MALDI analysis of
the J1-L2-OVA peptide treated in vitro with cathepsin B revealed
that the peptide is cleaved as predicted in the L2 region, again
supporting that the proteasome is not an absolute requirement for
epitope cleavage from the J1-L2-hybrid peptides (data not shown).
Interestingly, the TAP transporter was not absolutely required for
presentation of OVA from either HSP70:OVA-L1-J1 or HSP70:
J1-L2-OVA. There was some diminishment in cross-presentation
with HSP70:OVA-L1-J1 complex, however, there was no varia-
tion between C57BL/6 and TAPϪ/Ϫ
PECs in the ability to cross-
present OVA derived from HSP70:J1-L2-OVA complex (Fig. 4B).
In addition, lactacystin treated or untreated C57BL/6 PECs and
TAPϪ/Ϫ
PECs all exhibited similar abilities to present exog-
enously added OVA peptide (Fig. 4). These data indicate that
while the new J1-L2-epitope peptides may enter the proteasomal
pathway for processing and presentation, it is not an absolute re-
quirement, indicative of the epitopes being loaded into the MHC
class I peptide-binding groove by at least one other means.
The Javelin sequence is specific for HSP interaction and is
necessary for enhanced immunogenicity in response to HSP70:
Javelin hybrid peptide complexes
We next determined whether the optimized Javelin sequence (J1)
was required for the enhanced immunogenicity of the new J1-L2-
epitope constructs when injected in complex with HSP70. Mice
were immunized with HSP70 complexed with either J1-L2-OVA
or L2-OVA, and immune responses evaluated in the ex vivo
ELISPOT assay. As shown in Fig. 5A, there were no responses to
HSP70:L2-OVA above the negative control; however, there was a
good ex vivo response induced in mice immunized with HSP70:
J1-L2-OVA at an equimolar dose. There was likewise no response
in mice immunized with HSP70 alone (data not shown). These
results indicate that the linker alone does not confer the ability of
hybrid peptide to elicit immunity.
Although J1 improved immunogenicity with peptides in com-
plex with HSP, it was important to determine whether the effect
was specific to HSP or was a property of any large protein that can
act as a potential carrier. Mice were immunized with peptides com-
plexed with HSP70, or mixed with phosphorylase B or BSA under
the same conditions in which HSP70:peptide complexes are formed,
then evaluated for immunogenicity in the ex vivo ELISPOT. As
shown in Fig. 5B, there was minimal effect of peptides mixed with
BSA or phosphorylase B; however, there was a robust immune
response when the J1-L2-OVA peptide was immunized in com-
plex with HSP70. Taken together, these results show that peptides
FIGURE 4. Neither the proteasome nor the TAP
transporter are required for cross-presentation of
epitopes from HSP70:J1-L2-OVA complexes. Adherent
PEC from thioglycolate-induced mice were pretreated
for 1 h with 20 M lactacystin where applicable, pulsed
with 40 nM peptide in complex with 400 nM HSP70,
then cocultured with the B3Z T-T hybridoma as de-
scribed in Materials and Methods. Supernatant IL-2
quantities were measured by ELISA after 18 h. A,
C57BL/6 PECs untreated (Ⅺ) or lactacystin treated (■);
B, C57BL/6 (Ⅺ) or TAPϪ/Ϫ
PECs (■). IL-2 levels Ϯ
SD from triplicate wells evaluated in duplicate from one
representative experiment of at least six similar experi-
ments are shown.
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7. containing the Javelin sequence and the chaperone property asso-
ciated with HSP70 are necessary for the induction of enhanced
immune responses to defined epitopes.
Mice immunized with complexes of HSP70 and the new J1-L2-
OVA hybrid peptide are protected from tumor challenge
The new J1-L2-OVA peptide construct was additionally evaluated
for its ability to protect against tumor challenge in a prophylactic
model of tumor rejection. Mice were immunized on days 0 and 7
with HSP70:J1-L2-OVA or the appropriate controls and, on day
14, challenged in the flank with a s.c. injection of E.G7 cells (the
EL4 thymoma transfected with OVA cDNA). Tumor growth was
monitored every 3–4 days throughout the duration of the experi-
ment, as described in Materials and Methods. As shown in Fig. 6,
the mice immunized with either J1-L2-OVA alone or HSP70:J1-
L2-OVA complexes had significantly decreased tumor burden rel-
ative to the group immunized with diluent ( p Ͻ 0.02 and p Ͻ
0.001, respectively). Moreover, there were no differences between
the diluent group and any of the other immunized groups, includ-
ing the HSP70:OVA complex-immunized mice, indicating the ro-
bustness of the J1-L2-OVA vaccine. These data indicate that the
J1-L2- modification of epitopes not only increases their cross-pre-
sentation and ex vivo immunogenicity, but also renders the mod-
ified epitopes powerfully immunogenic in vivo in the presence and
absence of the HSP70 chaperone.
The J1-L2- sequence increases the affinity of several human and
murine epitopes for HSP70 resulting in robust immune
responses
To test whether the J1-L2-epitope hybrid peptide can be used to
deliver other MHC class I-binding epitopes in addition to OVA,
we synthesized constructs containing various defined murine and
human MHC class I-binding epitopes, and tested their ability to
induce immune responses in C57BL/6 and HLA-A*0201-trans-
genic HHDII mice, respectively. Table III shows the dissociation
constant (Kd) for each of the epitopes with and without the addition
of the Javelin linker. On average, there was a 68-fold increase in
affinity with the J1-L2- modification to each of the epitopes, ex-
cluding J1-L2-IMD, which had a 976-fold increase in affinity for
HSP70 compared with unmodified IMD (Table III). Mice were
immunized with the HSP70:J1-L2-epitope complexes and evalu-
ated for CD8ϩ
Ag-specific T cell responses in the ex vivo IFN-␥
ELISPOT assay. Fig. 7A shows the Ag-specific immune re-
sponses to three different murine H2-Kb
-binding antigenic
FIGURE 5. The Javelin sequence and HSP are required for the en-
hanced immunogenicity of HSP70:Javelin-peptide complexes. A, Mice
were immunized with HSP70:J1-L2-OVA or HSP70:L2-OVA or L2-OVA
alone (1.3 M protein:19 M peptide), then CD8-enriched splenocytes
were tested in an ex vivo ELISPOT assay 7 days postimmunization. Solid
bars, Effectors stimulated with an irrelevant epitope from vesicular stoma-
titis virus (VSV) in the absence of APC; open bars, effectors stimulated
with OVA in the absence of APC. B, Mice were immunized with 19 M
(2.5 g) J1-L2-OVA mixed with 1.3 M of the indicated proteins, or the
appropriate controls, and the immune responses measured by ex vivo
ELISPOT in the absence of APC. Data are the mean IFN-␥ spot number Ϯ
SE from three experiments. PhB, phosphorylase B.
FIGURE 6. Immunization with the complex of HSP70 and the im-
proved J1-L2-OVA construct provides protection against subsequent tumor
challenge. Ten mice per group were immunized s.c. base of the tail on days
0 and 7 then challenged with 7.5 ϫ 105
EG.7 cells on day 14, as described
in Materials and Methods. Mice were observed two to three times per week
for the presence of tumors and data are shown as the average tumor vol-
ume Ϯ SE for peptide only (16 M; solid symbols) or HSP70:peptide
complexes (1.1 M protein with 16 M peptide; open symbols). ,ء p value
Ͻ0.02; ,ءءء p value Ͻ0.001 by Dunnett’s method.
Table III. Affinities of multiple murine and human epitopes
Epitope MHC Source Ref.
Kd (M)
Epitope J1-L2-epitope
Bcas H-2 Kb
Bovine ␣1-caseinL1421-149 54 108 2.2
SdV H-2 Kb
Sendai virus NP324-332 55 131 2.4
Vsv H-2 Kb
Vesicular stomatitis virus NP52–59 56 84 1.0
YMD HLA-A*0201 Tyrosinase369-377 57 209 2.3
IMD HLA-A*0201 Modified gp100209-217, g209-2M 58 2567 2.6
Trp2 HLA-A*0201 Tyrosine-related protein-2180-188 59 82 1.2
MelA HLA-A*0201 Mart-127-3 60 46 1.2
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8. epitopes, J1-L2-Bcas, J1-L2-SdV, and J1-L2-Vsv. Substantial
IFN-␥ levels were secreted in response to all three epitopes
upon immunization with HSP70:J1-L2-epitope complexes.
Cells restimulated with irrelevant peptide had negligible spot
numbers (data not shown). Fig. 7B illustrates representative
data from HHDII-transgenic mice that were immunized with
complexes of HSP70 and HLA-A*0201-presented melanoma
hybrid epitopes J1-L2-YMD, J1-L2-IMD, J1-L2-Trp2, and J1-
L2-MelA. In all cases there was a high level of IFN-␥ produced
upon immunization with HSP70:J1-L2-epitope complexes. The
background spot number in response to irrelevant peptides in
these experiments was less than five spots per well (data not
shown). Taken together, these data indicate that the J1-L2- se-
quence can be used for several human and murine antigenic
epitopes to increase their affinity for HSP70, and form immu-
nogenic complexes.
Hybrid J1-L2-epitope peptides with uniform HSP70-binding
affinities allow multiple-epitope complexes to be immunized
within in a single vaccine
Creating hybrid peptides that shared similar affinities for HSP70
enabled us to immunize mice with more than one epitope in a
single injection. In theory, the similar HSP70-binding affinities re-
sulted in equimolar amounts of each peptide associated with
HSP70 at equilibrium, thus increasing the likelihood of generating
robust immune response to each peptide present in a multivalent
vaccine. To test this hypothesis, we immunized C57BL/6 or HLA-
A*0201 HHDII-transgenic mice with multiple-epitope vaccines.
Fig. 8A shows IFN-␥ ELISPOT data from mice that were immu-
nized with HSP70:J1-L2-OVA, HSP70:J1-L2-Vsv, or a complex
formulated with both peptides. Complexes were prepared as de-
scribed in Materials and Methods; where more than one peptide
was included in a complex the peptides were mixed together when
added to HSP70. The amount of HSP70 in each formulation was
the same. As observed previously, there was a response to the
J1-L2-OVA epitope immunized in the absence of HSP70. The in-
crease in observed immunogenicity of the J1-L2-OVA peptide in
Fig. 8A vs Fig. 5 is attributable to the increased dose of protein and
peptide immunized in Fig. 8A. J1-L2-Vsv alone did not stimulate
IFN-␥ production. The lack of response to this particular hybrid
peptide in the absence of HSP70 is not clear.
Also shown in Fig. 8A, there were substantial IFN-␥ responses
from mice immunized with HSP70:J1-L2-OVA or HSP70:J1-L2-
Vsv complexes; importantly, not only was there a robust response
when the mice were immunized with the two hybrid epitopes in a
single complex, there was also no diminishment in the responsive-
ness to either epitope when compared with the single complex
immunizations. Likewise, as shown in Fig. 8B, when complexes
containing human J1-L2-epitope melanoma Ags in association
with HSP70 were injected into the HHDII-transgenic mice, the
mice were able to generate responses to both epitopes. Coimmu-
nization of complexes that were prepared individually then mixed
just before immunization resulted in immune responses that mir-
rored the response to each epitope delivered in a single epitope
FIGURE 7. The J1-L2-modification is functional for several antigenic
epitopes. Mice were immunized s.c. base of the tail with peptides com-
plexed with HSP70 at the optimal concentrations determined for each pep-
tide. CD8-enriched splenocytes were evaluated 7 days later for peptide-
specific immune responses in the ex vivo ELISPOT assay without the
addition of naive APC. A, Murine hybrid epitopes immunized in complex
with HSP70 elicit immune responses in C57BL/6 mice. All data are shown
as the mean Ϯ SE of between 3 and 18 observations for each epitope. There
were no spots in irrelevant peptide wells (data not shown). B, Human
hybrid epitopes (5–10 g) immunized in complex with HSP70 (20–25 g)
elicit immune responses in HHDII HLA-A2.1-transgenic mice. Data are
shown as the mean Ϯ SD of four mice per group from one representative
experiment per peptide (from at least three observations per epitope). These
were negligible spots in unrelated peptide wells (data not shown). NT, Not
tested.
FIGURE 8. Ex vivo IFN-␥ ELISPOT responses to multiple epitopes in
a single immunization. A, C57BL/6 mice were immunized with controls,
single-epitope complexes, or two hybrid epitope complexes (in a single
injection), then tested for epitope-specific immune responses in the ex vivo
ELISPOT assay without the addition of naive APC. The HSP70 concen-
tration in each complex was 3.2 M. Open bars represent OVA-specific
responses, dark gray bars correspond to Vsv-specific responses, Light gray
bars show response to irrelevant peptide, and black bars are the medium
control wells. B, HHDII mice were immunized with single-epitope com-
plexes, a mixture of two single-epitope complexes (complex mix), or a
complex formulated with both epitopes at the same time (single complex);
7 days later, CD8 T cells were analyzed for epitope-specific IFN-␥ pro-
duction using peptide-pulsed naive splenocytes as APCs.
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9. complex, although the response to the IMD epitope was somewhat
diminished relative to that peptide immunized in a single epitope
complex. In contrast, when the two epitopes were mixed when
complexed with HSP70, there was an “equalized” immune re-
sponse such that the response to the YMD was augmented over the
response to the same epitope immunized in a single epitope
complex.
Discussion
The modification of antigenic epitopes by collinear synthesis with
a high-affinity HSP-binding sequence enhances their immunoge-
nicity (34). As shown here, optimization of the Javelin sequence
further increases and equalizes the affinity of antigenic epitopes for
HSP70. Additionally modifying the linker sequence to include pu-
tative cleavage sites and changing the orientation of the construct
to the N terminus of the epitopes resulted in added enhancement of
immunogenicity. Moreover, this peptide modification is univer-
sally applicable to both murine and human epitopes, enhancing the
likelihood of efficacy of vaccines containing multivalent, HSP70:
peptide complexes.
There is a strong body of literature that illustrates how HSP70
and other cell-derived chaperones can serve as adjuvants to deliver
antigenic epitopes to the host immune system and induce cell-
mediated immunity against tumor and viral targets (10–15, 17–20,
34, 46, 47). These are very crucial observations because histori-
cally, vaccines delivered with conventional adjuvants designed to
elicit T cell responses have been poorly tolerated or plagued with
side effects that have restricted their approval for human use (48).
However, there are potential limitations to vaccines made by load-
ing HSPs with diverse peptides in vitro. Individual peptide se-
quences have quite variable affinities for HSP (33, 34), and in
equilibrium reactions in which more than one peptide is com-
plexed with HSP70 in vitro, a higher affinity peptide will bind HSP
at the expense of the lower affinity peptide, with the potential effect
of inefficient priming of the immune system to the underrepre-
sented peptide.
It was previously demonstrated that hybrid peptides containing
an antigenic epitope collinearly synthesized with J0, a high-affinity
HSP70-binding sequence, complexed with HSP70 could dramati-
cally enhance the potency of immunization when compared with
the unmodified epitope complexed with HSP70 (34). The in-
creased efficacy of immunization was attributed to improved af-
finity of the peptides for the HSP70. However, evaluation of the
binding kinetics of a variety of the J0-hybrid peptide constructs
revealed that while the dissociation constants were lowered 5- to
30-fold relative to the minimal epitope alone, the epitopes still
greatly influenced the binding affinity of the J0-hybrid peptides.
The binding affinities of the J0-hybrid peptides reported here cover
two orders of magnitude (4.2–180 M; Table II). These differ-
ences could affect the ability of multiple epitopes to be delivered
equally in a single vaccination.
Modifying the Javelin to an even higher affinity HSP-binding
sequence (J1) normalizes the HSP70 affinity of all the J1-hybrid
peptides tested such that their dissociation constants are within
ϳ3 M of one another, thus creating a more “universal” Javelin
(Tables II and III; Fig. 1). The higher affinity interaction of any
given peptide with HSP70 results in a better chance for the
HSP-complexed peptide to be delivered to APCs with high effi-
ciency resulting in robust immune responses. Moreover, the higher
affinity sequence resulted in greater uniformity of responses be-
tween experiments. In addition, combining multiple peptides that
have been modified with the J1 sequence with HSP70 makes it
possible to deliver more than one epitope in a single injection with
similar efficiencies (Fig. 8).
Evaluation of the new Javelin (J1)-hybrid peptide led to the
conclusion that the higher affinity Javelin sequence was an im-
provement over the previously published Javelin (J0) constructs.
Nevertheless, the frequency of IFN-␥-secreting CD8ϩ
T cells in-
duced by immunization with HSP70:J1-hybrid peptide complexes
was rather low (Fig. 2B, 1 cell/10,000), indicating less-than-opti-
mal immune responses. This observation led to additional reeval-
uation of the J1-hybrid peptide in an attempt to further improve the
immunogenicity of the HSP70:Javelin-peptide complexes.
Data from Castellino et al. (21) using J0-modified peptides re-
vealed that both cytosolic and endocytic routes were responsible
for MHC class I presentation of the HSP-associated peptides. The
orientation of the Javelin-peptide sequence governed the observed
effect, such that when the J0 was synthesized C-terminal to the
epitope, the peptide was processed via the cytosolic route, but
when the J0 was synthesized N-terminal to the epitope, the peptide
was processed via the endocytic route. One of the explanations for
the difference may be the affinity of the peptide for HSP70. OVA-
L1-J0 has ϳ16-fold lower affinity for HSP70 than J0-L1-OVA
(P. Slusarewicz and A. Kays Leonard, unpublished observations).
The difference in affinity has at least three implications. First, the
literature reveals that peptide binding changes the conformation
and rigidity of HSP70 (49, 50), leaving the possibility that higher
affinity peptides may have a more profound affect on the HSP70
conformation, potentially altering which receptor the HSP-peptide
complex binds and as a result becomes incorporated into the APC.
Second, a higher affinity interaction may affect how a peptide is
processed within the cell by protecting the peptide from proteolytic
digestion thereby increasing its half-life within the cell (51). Or
third, the result may be that a greater proportion of HSP70 is bound
by peptide, and at least for the CD40 receptor, the interaction with
HSP70 is strongly increased when HSP70 is complexed with a
peptide substrate (31). However, it seems the affinity is not the
only explanation for route of processing, because OVA-L1-J1 and
J1-L2-OVA have similar affinities for HSP70, yet OVA-L1-J1 is
dependent on the proteasome for processing, but J1-L2-OVA is not
(Fig. 4). Because the proteasome is responsible for correct C-ter-
minal cleavage of epitopes (reviewed in Ref. 52), and the peptide
J1-L2-OVA already has the correct C terminus but OVA-L1-J1
does not, it is possible that the difference observed is simply due to
the requirement for processing. The data do not rule out that J1-
L2-OVA can enter the cytosolic pathway of peptide processing.
Rather, they suggest that the proteasome is not an absolute require-
ment for MHC class I presentation of this peptide.
Further evidence that peptide processing is also significant for
increased immunogenicity is illustrated by comparing J1-L1-OVA
and J1-L2-OVA. These two peptides share the Javelin-epitope ori-
entation and the concomitantly low HSP70-binding affinities of the
J1 epitope (1.63 vs 2.26 M, respectively), yet J1-L2-OVA in-
duces immune responses greater than predicted by the difference in
the binding affinities (Fig. 3). Indeed, the increase in immune re-
sponses must be attributed to the linker modification, because all
other components are the same. The L2 linker sequence created an
optimal target for constitutively expressed proteolytic enzymes as
well as a potential cleavage site for the proteasome itself, thereby
increasing the potential for the correct epitope sequence to be gen-
erated regardless of peptide delivery into the cytosolic or endocytic
routes of peptide processing.
The optimized Javelin-linker-epitope sequence is quite potent,
and it is interesting to note that the Javelin-modified peptides have
weak to moderate immunizing potential on their own (Figs. 6–8),
repeating a phenomenon observed with the OVA-L1-J0 peptide
(34). Experiments are currently underway to understand how the
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10. peptides can be immunogenic in the absence of HSP70 or adju-
vant, but there are several working hypotheses. First, the peptide
may be binding endogenous HSP70 that is released as a result of
cellular damage that is incurred upon immunization or present in
the serum of individuals. Indeed, the peptide induces better im-
mune responses in the presence of ADP (J. B. Flechtner, unpub-
lished observations), suggesting that this may be the case because
ADP enhances HSP70-peptide interactions. Second, the hydropho-
bic nature of the peptide may render it a cell-penetrating peptide,
such that it is inserted into the cytoplasmic membrane of the cell
and internalized. Third, again due to the hydrophobic nature of the
Javelin sequence, the peptide may form aggregates or other higher-
order structures in the absence of HSP and therefore be taken up
into APCs by phagocytosis. Regardless of the mechanism, immu-
nization of mice with J1-L2-OVA in saline is quite effective at
protecting them against a tumor challenge. It is surprising, there-
fore, that there can be such low numbers of CD8 T cells producing
IFN-␥ in response to the J1-L2-OVA peptide immunized in the
absence of HSP70 (Fig. 5B) but robust antitumor responses (Fig.
6). This may be a direct reflection of the relative insensitivity of the
ELISPOT to predict effector T cell efficiency, or more likely, a
reflection of increase in CTL frequency that occurs as a result of
the prime-boost regimen used in the tumor rejection assay com-
pared with the prime-only protocol used for the ex vivo ELISPOT
analysis. It will be of interest to test whether under conditions of
limited peptide (Ͻ16 M in the experiment described in Fig. 6),
the J1-L2 modification renders peptides delivered in complex with
HSP more immunogenic than the corresponding amounts of un-
modified peptide in complex with HSP.
Importantly, robust immune responses to peptides modified with
the Javelin-linker sequence are not limited to a single epitope. Both
human and mouse antigenic epitopes are amenable to modification
with the Javelin sequence and remain potent stimulators of the
immune system when delivered in complex with HSP70. Interest-
ingly, the Trp2 Ag is a “self” epitope for both mice and humans
that typically requires multiple immunizations to break tolerance
to the Ag (53). However, in the HHDII mice, only one immuni-
zation was required to break tolerance and induce strong immune
responses.
Finally, the Javelin modification allows more than one epitope
to be delivered in a single immunization. Notably, there does not
appear to be an immunodominance issue–multiple epitopes can be
delivered in a single vaccine without great expense to the response
to an individual epitope. Currently, work is in progress to deter-
mine whether a single Javelin-linker sequence can be used to mod-
ify a “string” of several epitopes to circumvent potential solubility
issues with the relatively hydrophobic Javelin sequence.
In summary, modifying the sequence, linker, and orientation of
Javelin-epitope constructs not only increases and normalizes their
affinity for HSP70 but optimizes their ability to be delivered to
APCs, processed and presented by the cell, and in turn induce
robust immune responses. The Javelin-hybrid peptides can be
mixed together and complexed with HSP70 resulting in multiva-
lent immune responses that are as potent as immunizing separate
HSP70/peptide complexes that are mixed just before immuniza-
tion. Thus, with the new Javelin-linker modified peptides, the el-
egantly characterized, adjuvant-free, HSP-based vaccine is opti-
mized to deliver multiple Ags with equal efficiency to induce
potent immune responses to either infectious disease or tumor
targets.
Acknowledgments
We are grateful to the former crew of Mojave Therapeutics, especially
Priscilla Calderon, Armin Lahiji, Kevin Wright, Adrienne Scott, and Cara
Miller for technical assistance with immunological assays, as well as Na-
dine Soplop, Nicole Covino, George Angelos, Jeff Courter, and Jason Ten-
zer for technical assistance with biochemical assays. Thanks to Denise
Ireland, Jennifer Burke, and the crew of the animal facility at Antigenics
for experimental help and animal handling. We also thank Nilabh Shastri
for the B3Z T-T hybridoma, Franc¸ois Lemmonier for supplying the HH-
DII-transgenic mice, and Roman Chicz and Robert Binder for critically
reading this manuscript.
Disclosures
J. B. Flechtner, K. P. Cohane, S. Mehta, P. Slusarewicz, B. H. Barber, and
S. Andjelic have two pending patents, both titled “Improved heat shock
protein-based vaccines and immunotherapies.” Both patents were filed by Mo-
jave Therapeutics, and the Intellectual Property was assigned to Antigenics
Inc. D. L. Levey is a current, stockholding employee of Antigenics Inc.
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