Supplementary Table 2. Induction and tolerization of atherosclerosis with heat shock proteins and their reactive T-cell epitopes in experimental animals
Immunization - Induction of disease
HSPs
Position
Amino acid sequence
Animal model
Administ
Outcome
Ref
ration
mHSP65
GroEL
153-171
153-171
DQSIGDLIAEAMDKVGNEG
DETVGKLIAEAMDKVGKEG
PgHSP60p
PgHSP60p
NP
NP
TVPGGGTTYIRAIAALEGLK
TLVVNRLRGSLKICAVKAPG
hHSP60
437-450
448-460
437-460
VLGGGCALLRCIPA
IPALDSLTPANED
VLGGGCALLRCIPALDSLTPANE
D
s.c.
Induces in vitro proliferation of peptide or mHSP65-primed cells.
Elicited strong immunogenic properties after immunization with this
peptide.
1
s.c.
2
C57BL/6J
s.c.
Monoreactive monoclonal antibody to PgHSP60 recognized peptide
(TVPGGGTTYIRAIAALEGLK), while polyreactive monoclonal antibody
recognized peptide (TLVVNRLRGSLKICAVKAPG). The cross-reactivity
was confirmed based on known sequences of the human HSP60
molecule as well as lysates from heat-induced whole bacterial cellular
protein. Thus, this data suggests that this peptide may be an
immunodominant epitope with a robust cross-reactivity with other
bacterial and human HSP60s.
Immunization with “P277” (VLGGGCALLRCIPALDSLTPANED) caused a
marked increase in vascular leak syndrome (VLS). B-cell epitopes
located in “P277” played a causal role in the development of VLS.
Moreover, anti-P277 antibodies could cross-react with HSP60, highly
expressed in both normal and stressed ECs, and mediate damage to
cells in the presence of complement. These data suggested that a
humoral immune response induced by anti-P277 immunity mediates
EC damage and induces VLS.
Tolerization - Treatment of disease
Animal model
Administ
ration
3
HSPs
Position
mHSP60
mHSP60p
HSP70*
hApoB /+/
hHSP60
Full-length
253-268
111-125
688-707
/+/
153-163
Full-length
EGEALSTLVVNKIRGT
ITDAVITTPAYFNDA
IEIGLEGKGFEPTLEALFGK
AELKKQSKPVT
LDLr-/-
Oral
Increased number of Tregs, IL-10, and TGF-β. Decreased aortal lesion
formation.
4
ApoBtm25gyLDLrtm1Her
RIMMS
5
hHSP60
ApoB
153-163
661-680
AELKKQSKPVT
IEIGLEGKGFEPTLEALFGK
ApoBtm25gyLDLrtm1Her
Oral
hHSP60
153-163
AELKKQSKPVT
ApoBtm25gyLDLrtm1Her
Oral
IEIGLEGKGFEPTLEALFGK
AELKKQSKPVT
ApoBtm25gyLDLrtm1Her
RIMMS
Immunization with a combination of ApoB and HSP60 peptide antigens
significantly reduced early atherosclerotic lesions. In addition, high
levels of ApoB and HSP60 antibodies were detectable after
immunization.
Increased expression of TGF-β, CD4+CTL-4+, TNF-α, MMP-9 and number
of macrophages and Tregs. Decreased aortal lesion formation and
increased plaque stabilization.
Induced a T-cell proliferation and expansion of Tregs with IL-10 and
TGF-β. Moreover, it reduced early atherosclerotic lesion formation.
Immunization of mice with a single construct containing multiple
epitopes derived from ApoB100, hHSP60, and Chlamydia pneumonia
hApoB /+/
hHSP60
688-707
/+/
Amino acid sequence
BALP/c, CBA,
C57BL/10, B10.D2,
B10.BR, B10.M,
B10.S, B10.A
C57BL/6J
Outcome
Ref
6
7
8
/+/
hHSP60
/+/
MOMP /+/
Cp
mHSP65
mHSP65
mHSP65
mHSP65
153-163
/+/
303-312
/+/
67-74 /+/
283-291
91-105
106-125
111-125
312-326
PGFGDNRKNQ
GDYVFDRI
QAVANGGAI
ATVLAQALVREGLRN
VAAGANPLGLKRGIEKAVEK
NPLGLKRGIEKAVEK
LSLLGKARKVVVTKD
was more effective in reducing early atherosclerotic lesions through
the induction of a specific Treg-cell response than was the construct
containing either mono- or bi-epitopes.
New Zealand white
rabbits + C57BL/6J +
LDLr-/-
s.c.
Atherosclerosis was significantly enhanced in HCD fed immunized
rabbits. Immunized mice had increased aortic endothelial injury.
Although western blot demonstrated that specific antibodies against
mHSP65(91-105) can cross-react with recombinant hHSP60, specific
antibodies against mHSP65(91-105) had no direct effects on HUVECs in
vitro, even when the HUVECs were heat shocked. Adoptive transfer of
mHSP65(91-105) specific splenic cells can accelerate atherosclerosis in
LDLr-/- mice.
9
s.c.=subcutaneous, RIMMS=The repetitive immunization multiple sites strategy, Cp=Chlamydia pneumonia, PgHSP60=Porphyromonas gingivalis HSP60, HCD=highcholesterol diet, NP=Not performed.
*=Sequence was based on a partially conserved human, rat, and mouse sequence of the HSP70 molecule.
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