Transcript Functional Microbial Genomics

Functional Microbial
Genomics…HIV
Shainn-Wei Wang, Ph.D.
NCKU, College of Medicine
Institute of Molecular Medicine
HIV exhibits tremendous genetic diversity
Garber D. A., et al., Lancet Infec. Dis., 2004
HIV Data Base
• http://www.hiv.lanl.gov/content/index
HIV/HCV Database
http://lasp.cpqgm.fiocruz.br/LaspIngles/ferramentas.html
http://www.flu.lanl.gov/
Human Immunodeficiency Virus (HIV)
The nature of HIV infection
Persistent and chronic infection
- venereal spread (mucosal immunity)
- hematogenous spread (systemic immunity)
- poor immunogenicity and viral escape
- progressive destruction (impaired regeneration of CD4 T help)
Acute
infection
Latent
infection
Infection and reservoir
Functional genomics of HIV
infection
• Host gene re-programming to viral infection
– Host cells or Immune cells related
– Immune suppression or activation
– Invasion or evasion
• Transcriptional networks in response to a viral
protein
• Cellular protein networks in response to
assembly, replication, and latency
• Functional search for MHC epitopes that are
related to disease protection or progression
• Epitope based vaccines
Mock control
PBMC
Target cells
Non-Infection: gene sets
Infection: signature gene sets
comparison
Change in disease or functional Status
The Journal of Infectious Diseases
2004;189:572-582
Functional genomics of HIV
infection
• Host gene re-programming to viral infection
– Host cells or Immune cells related
– Immune suppression or activation
– Invasion or evasion
• Transcriptional networks in response to a viral
protein
• Cellular protein networks in response to
assembly, replication, and latency
• Functional search for MHC epitopes that are
related to disease protection or progression
• Epitope based vaccines
Reprogramming the iDC
• As the major activator of HIV
transcription, Tat drives viral gene
expression.
• Tat regulates the expression of
chemokines that promote lymphocyte
and monocyte migration.
• By recruiting susceptible host cells to
infected dendritic cells, Tat may
facilitate HIV dissemination
Expression analysis of primary iDC infected with HIV-1BAL and adeno-Tat
- Expression profiles of iDC genes whose RNA levels were affected similarly by adeno-Tat and
HIV-1 infection.
- Genes are divided into functional groups; the fold change in expression levels relative to the
0 time point is displayed in red (increased expression) or green (decreased expression).
- Asterisk (*) marks IFN-inducible genes.
What they found in the array:
• HIV-1 Tat induces expression of
interferon-inducible genes
• HIV-1 Tat mediates chemokine
upregulation in iDC
RT-PCR analysis of selected immature dendritic cell
genes whose expression is affected byTat.
• Identical amounts of total RNA from
iDC infected with adeno-LacZ and
adeno-Tat were used.
• Control β-actin mRNA was not
affected by HIV-1 or adeno-Tat,
confirming the microarray analysis.
Rt-PCR gene expression analysis in iDC obtained from donors
A, B, C,D: donors
MCP-2 expression and SIV infection in axillary lymph nodes
a, SIV Nef–expressing cells (red) in
the paracortex.
b, Digital overlay of images
from the same field labeled for MCP-2
(green) and SIV Nef (red). Arrows
indicate double-labeled cells (yellow)
that are positive for both markers in
the digital overlay of images.
c–e, High-powered fields of lymph
nodes showing SIV Nef (c), MCP-2 (d)
and DC-SIGN (e, blue) expression by
a single cell (indicated by arrows).
f, Digital overlay of c–e shows a
single SIV-infected dendritic
cell (indicated by arrow) expressing
all three markers (original
magnification: a,b, ×200, c–f, ×400).
Chemotaxis of Monocytes and activated T cells
None of the typical dendritic cell maturation markers
(such as CD40, CD80, CD83, CD86 and CD25) were
expressed at increased levels during the time course of
adeno-Tat, adeno-LacZ or HIV-1 infection
Summary and Discussion of the results
• Genes encoding four different chemokines were induced
in iDC by both Tat expression and HIV-1 infection
–
–
–
–
(interferon inducible protein-10 (IP-10),
human monokine induced by interferon-γ (HuMIG),
monocyte chemoattractant protein-2 (MCP-2)
monocyte chemoattractant protein-3 (MCP-3)
• IP-10 and HuMIG are CXC chemokines whose
production is normally IFN-γ dependent. These two
chemokines attract activated T cells, whose chemotaxis
is mediated by the CXCR3 receptor.
• MCP-2 and MCP-3 are CC chemokines that attract
monocytes and are produced by a variety of cells,
including fibroblasts, endothelial cells, monocytes and
macrophages
• HIV-1 infection and Tat expression do not result in
activation and maturation of iDC
– No production of cytokines that are a hallmark of dendritic cell
activation,
– No phenotypic changes typical of dendritic cell maturation
– the induction of IP-10, HuMIG, MCP-2 and MCP-3 in iDC by Tat
and HIV-1 seems especially important for the spreading of HIV
• The lack of iDC maturation with respect to cell surface
markers paralleled the lack of induction of
proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL10, IL-12)
– whether the lack of iDC maturation after HIV-1 infection adversely
affects the adaptive immune response require further exploration
confirmation
• Therapies designed to affect Tat function may produce
the combined benefit of limiting viral transcription and
reducing the interactions between infected dendritic cells
and T cells that contribute to the expansion of viral
infection
Genomic Database for HIV Infection
Functional genomics of HIV
infection
• Host gene re-programming to viral infection
– Host cells or Immune cells related
– Immune suppression or activation
– Invasion or evasion
• Transcriptional networks in response to a viral
protein
• Cellular protein networks in response to
assembly, replication, and latency
• Functional search for MHC epitopes that are
related to disease protection or progression
• Epitope based vaccines
Hepatocyte-growth factorRegulated tyrosine kinase
Substrate
The Protein Network of HIV Budding, Cell, Vol. 114, 701–713, September 19, 2003
Proteome analysis
• Protein-Protein interaction
– Two Hybrid Sysytem
– (Tandem) Affinity Tag
• Protein complex purification requires expression of the
target protein at, or close to, its natural expression level.
• Among all kinds of protein complex purification
method, protein A and CBP tags allowed efficient
recovery of proteins
• Proteome analysis, in particular using mass
spectrometry (MS), requires fast and reliable methods of
protein purification.
Tandem Affinity Purification Method
Guillaume Rigaut, 1999
TEV cleavage site
calmodulinbinding peptide
TAP tag
protein A
Tandem Affinity Purification Method
Protein composition of TAPpurified U1 snRNP.
Guillaume Rigaut, 1999
Tandem Affinity Purification Method
Guillaume Rigaut, 1999
Tandem Affinity Purification Method
Protein composition
of TAP-purified U1
snRNP.
Guillaume Rigaut, 1999
Tandem Affinity Purification Method
Protein composition
of TAP-purified U1
snRNP.
Guillaume Rigaut, 1999
Protein identification and
functional analysis
MALDI-TOF
Protein samples
from GE
- Online Data mining
- Functional assay
Nature. 2002 Jan 10;415(6868):141-7.
Synopsis of the screen
a. Schematic representation of the gene targeting procedure The TAP cassette is inserted at the C terminus of a given
yeast ORF by homologous recombination, generating the TAP-tagged fusion protein.
b. Examples of TAP complexes purified from different subcellular compartments separated on denaturing protein gels
and stained with Coomassie. Tagged proteins are indicated at the bottom. ER, endoplasmic reticulum.
c, Schematic representation of the sequential steps used for the purification and identification of TAP complexes (left),
and the number of experiments and success rate at each step of the procedure (right).
The polyadenylation machinery
Primary validation of complex composition by `reverse' purification:
a, A similar band pattern is observed when different components of the
polyadenylation machinery complex are used as entry points for affinity
purification. Underlined are new components of the polyadenylation machinery
complex for which a physical association has not yet been described. The bands
of the tagged proteins are indicated by arrowheads.
b, Proposed model of the polyadenylation machinery.
The protein complex network, and
grouping of connected complexes
Links were established between complexes sharing
at least one protein. For clarity, proteins found in
more than nine complexes were omitted. The
graphs were generated automatically by a
relaxation algorithm that finds a local minimum in
the distribution of nodes by minimizing the
distance of connected nodes and maximizing
distance of unconnected nodes. In the upper panel,
cellular roles of the individual complexes are
colour coded: red, cell cycle; dark green, signalling;
dark blue, transcription, DNA maintenance,
chromatin structure; pink, protein and RNA
transport; orange, RNA metabolism; light green,
protein synthesis and turnover; brown, cell polarity
and structure; violet, intermediate and energy
metabolism; light blue, membrane biogenesis and
traffic. The lower panel is an example of a complex
(yeast TAP-C212) linked to two other complexes
(yeast TAP-C77 and TAP-C110) by shared
components. It illustrates the connection between
the protein and complex levels of organization. Red
lines indicate physical interactions as listed in
YPD22.
Functional genomics of HIV
infection
• Host gene re-programming to viral infection
– Host cells or Immune cells related
– Immune suppression or activation
– Invasion or evasion
• Transcriptional networks in response to a viral
protein
• Cellular protein networks in response to
assembly, replication, and latency
• Functional search for MHC epitopes that are
related to disease protection or progression
• Epitope based vaccines
CTL activity
HIV encoded proteins
MHC
CD4 # or function
HIV viral load
- Viral genetic diversity
- individuals infected with different viral clades
show different response patterns
- MHC genetic variability in human populations
Caucasian
Africans
Asians
Hispanics
South American
Indians
Disease Protection or
Disease progression
- these populations differ in their HLA allele
frequencies
- the HIV-specific responses detected in
these ethnicities can also differ
significantly.
HLA molecules may be directly involved
in restricting HIV replication
• The human leukocyte antigens (HLAs) are also known as
MHC (major histocompatiblity complex) or "self" molecules.
• T and B cells recognize antigens only when "presented" to
them next to an MHC ("self") molecule.
• There are two main types of HLA.
– Class I is divided into HLA A, B, C and are expressed by most
human cells.
– Class II is divided into HLA DP, DQ, DR and are expressed by B,
macrophages and dendritic cells. Class II is involved in presenting
antigen to CD4 cells, thus activating CD4 cells. When CD4 cells
recognize antigen presented by HLA class II, they secrete cytokines
(e.g., IL-2, IL-4) which in turn stimulate further immune responses
Nat Med. 2005 Dec;11(12):1290-2. Epub 2005 Nov 20
The epitopes tested are described in the Los Alamos database (www.hiv.lanl.gov
contentimmunologytablesctlsummary.html) in the context of HIV infection and are
listed in Table 2
The frequency of HLA supertypes within the cohort is negatively
correlated with the supertype capacity to elicit CTL responses.
The HLA alleleic variants that bind peptides possessing a particular HLA
supermotif are collectively referred to as an HLA supertype
(Sette and Sidney 1998; Sidney et al. 1996a, b, c).
Discussion
•
•
HLA class I alleles B27 and B57 are associated with better disease
prognosis, while others (such as B35) are associated with worse outcome
Other alleles like A3 and A11, which fall in the same HLA supertype (A3),
are differentially associated with disease progression, despite the fact that
they share multiple CTL epitopes.
•
Thus, in this case, the HLA molecule itself may have a more pronounced
influence on disease progression than the epitope
•
However, the role of the presented epitope and the restricting HLA allele in
this disease progression is still unclear
•
Future research should focus on the assessment of the functional avidity of
the epitope-specific T cell receptor (TCR) for the given HLA/peptide
complex and epitope binding affinity to the respective restricting class I
molecule.
• Potential epitope vaccines need to be tailored not only based on
local viral sequence diversity, but also on the local HLA allele
distribution.
In searching HIV and HCV
antigens involved in protective
immunity.
Published 22 Env-specific Immunodominant CD4 T epitopes based on HCV 77
HVTNDCPNSSIVYEA
E1
202-216
CPNSSIVYEAHDAIL
E1
207-221
SRCWVALTPTLAARN
E1
236-250
RCWVAMTPTVATRDG
E1
237-251
MTPTVATRDGKLPAT
E1
242-256
KLPATQLRRHIDLLV
E1
252-266
TIRRHVDLLVGAAAF
E1
257-271
GSATLCSALYVGDLC
E1
267-281
GSVFLIGQLFTFSPR
E1
282-296
TFSPRRHQTVQDCNC
E1
292-306
SIYPGHITGHRMAWD
E1
307-321
HITGHRMAWDMMMNW
E1
312-326
HVSGHRMAWDMMMNW
E1
312-326
HVSGHRMAWDMMMNWA
E1
312-327
MMMNWSPTAALVMAQ
E1
322-336
SPTAALVVSQLLRIP
E1
327-341
QAILDMIAGAHWGVL
E1
342-356
HWGVLAGLAYYSMVG
E1
352-366
FSMVGNWAKVLVVLL
E1
362-376
NWAKVLVVLLLFAGV
E1
367-381
GFATQRLTSLFALGPSQK
E2
393-410
CLWMMLLIAQAEQALL
E2
734-749
Published 22 Core-specific Immunodominant CD4 T epitopes
based on HCV 77 sequence
Epitope
Protein H77 Location Species HLA
MSTNPKPQIKTKRNTNRR
Core
1-18
human
MSTNPKPQRKTKRNTNRRQD
Core
1-20
human
DVKFPGGGQIVGGVYLLPRR
Core
21-40
human
DRB1*1101,
DQB1*0301
VGGVYLLPRR GPRLG ?
Core
31-45
human
DR11
VGGVYLLPRRGPRLGVRATR
Core
31-50
human
KVIDTLTCGFADLMGYIPLV
Core
121-140
mouse
H-2r, H-2b, H-2d
GAPLGGAARA LAHGV ?
Core
141-155
human
DR11, DR12
GAPLGGAARALAHGVRVLED
Core
141-160
human
APLGGAARALAHGVR
Core
142-156
human
SFSIFLLALLSCLTI
Core
173-187
human
LLSCLTIPASA
Core
181-191
human
DR
Published 22 Env-specific Immunodominant CD4 T epitopes
based on HCV 77 sequence
HVTNDCPNSSIVYEA
E1
202-216
human
CPNSSIVYEAHDAIL
E1
207-221
human
SRCWVALTPTLAARN
E1
236-250
human
RCWVAMTPTVATRDG
E1
237-251
human
MTPTVATRDGKLPAT
E1
242-256
human
KLPATQLRRHIDLLV
E1
252-266
human
TIRRHVDLLVGAAAF
E1
257-271
human
GSATLCSALYVGDLC
E1
267-281
human
GSVFLIGQLFTFSPR
E1
282-296
human
TFSPRRHQTVQDCNC
E1
292-306
human
SIYPGHITGHRMAWD
E1
307-321
human
HITGHRMAWDMMMNW
E1
312-326
human
HVSGHRMAWDMMMNW
E1
312-326
human
HVSGHRMAWDMMMNWA
E1
312-327
mouse
MMMNWSPTAALVMAQ
E1
322-336
human
SPTAALVVSQLLRIP
E1
327-341
human
QAILDMIAGAHWGVL
E1
342-356
human
HWGVLAGLAYYSMVG
E1
352-366
human
FSMVGNWAKVLVVLL
E1
362-376
human
NWAKVLVVLLLFAGV
E1
367-381
human
GFATQRLTSLFALGPSQK
E2
393-410
human
CLWMMLLIAQAEQALL
E2
734-749
human
DRB1*1101
Published Env-specific Immunodominant CD4 T epitopes
based on HXB2 sequence
DTEVHNVWATQACVPTDPNP
gp160
62-81
human
CVPTDPNPQEVV
gp160
74-85
human
NPQEVVLVNTENFNMWKND
gp160
80-99
human
YFNMWKNNMV
gp160
92-101
human
YFNMWKNNMVDQMHEDIISL
gp160
92-111
human
EQMHEDIISLWDQSLKPCVK
gp160
102-121
human,
mouse
HEDIISLWDQSLK
gp160
105-117
human
HEDIISLWDQSLKPCVKLT
gp160
105-123
human
IISLWDQSLKPC
gp160
108-119
human
SLWDQSLKPCVKLTPL
gp160
110-125
human
WDQSLKPCVKLTPLCVSLK?
gp160
112-130
human
SLKPCVKLTPLC
gp160
115-126
human
SLKPCVKLTPLCVSL
gp160
115-129
human
TTSNGWTGEIRKGEIKNCSF
gp160
138-159
human
KNCSFNISTSIRGKV
gp160
155-169
human
RDKMQKEYALLYKLDIVSID
gp160
166-185
human
DR
H-2k, H-2s
HLA-DR
HLA-DR
SVITQACSKVSFE
gp160
199-211
human,
chimpanzee
VITQACPKVSFEPIP
gp160
200-214
human
HLA-DR
SFEPIPIHYCAP
gp160
209-220
human
DR
PIPIHYCAPAGFAILKCNNK
gp160
212-231
human
PAGFAILKCNNKTFN
gp160
220-234
human
PAGFAILKCNNKTFNY
gp160
220-235
human
DR2
FAILKCNNK
gp160
223-231
human
DR2,6
NKTFNGKGPCTNVSTY
gp160
230-245
human
TNVSTVQCTHGRPIY
gp160
240-255
human
GIRPIVSTQLLLNGSC
gp160
250-265
human
EVVIRSANFTDNAKT
gp160
269-283
human
VVIRSDNFTNNAKTIC
gp160
270-285
human
SANFTDNAKTIIVQL
gp160
274-288
human
NFTDNAKTIIVHLNESVQIN
gp160
276-295
human
NAKTIIVQLNESVAIC
gp160
280-296
human
NESVAINCT
gp160
289-297
human
SVVEINCTRPNNNTRKS
gp160
290-306
human
VEINCTRPNNNTRKRIRIQ?
gp160
292-310
human
RIHIGPGRAFYTTKN
gp160
308-322
human
RIQRGPGRAFVTIGK
gp160
308-322
human
DR
RIHIGPGRAFYTTKNIIGIT
gp160
308-327
human
DRB1*0101
EQRGPGRAFVTIGKI
gp160
309-323
human
IQRGPGRAFVTIGKIGN
gp160
309-325
human
GRAFVTIGKIGNMRQ
gp160
314-328
human
RIIGDIRKAHCNISRY
gp160
321-336
human
CNISRAQWNNTLEQI
gp160
331-345
human
TLEQIVKKLREQFGNC
gp160
341-356
human
QIVKKLREQFGNNK
gp160
344-357
human
QSSGGDPEIV
gp160
363-372
human
SSGGKPEIVTHSFNC
gp160
364-378
human
PEIVTHSFNCGGEFF
gp160
369-383
human
GEFFYCNSTQLFNS?
gp160
380-393
human
EFFYCNTTQLFNNTW
gp160
381-395
human
FNNTWRLNHTEGTKGC
gp160
391-405
human
TWFNSTWSTKGSNNT
gp160
394-408
human
TWSTKGSNNTEGSDT
gp160
399-413
human
GSDTITLPCRIKQFINMWQE
gp160
410-429
human
LPCRIKQIINMWQEVY
gp160
416-431
human
CRIKQIINMWQGVGKAMYA
gp160
418-436
human, chimpanzee
KQIINMWQEVGKAMYA
gp160
421-436
human
FINMWQEVGKAMYAPPIS
gp160
423-440
human
INMWQEVGKAMYAPP
gp160
424-438
human
MWQEVGKAMYAPPIGC
gp160
426-441
human
APPIGGQISCSSNITY
gp160
436-451
human
IGGQIRCSSN
gp160
439-448
human
SSNITGLLLTRDGGTC
gp160
446-461
human
RDGGTNVTNDTEVFRC
gp160
456-470
human
GNSNNESEIFRPGGG
gp160
459-473
human
FRPGGGDMRDNWRSEL
gp160
468-483
human
DMRDNWRSELYKYKV
gp160
474-488
human
YKYKVVKIEPLGVAP
gp160
484-498
human
KYKVIKIEPLGIAPTC
gp160
485-500
human
TKAKRRVVEREKR
gp160
499-511
human
TKAKRRVVQREKRAAIGALF
gp160
499-519
human
GIVQQQNNLLRAIEA
gp160
547-561
human
QQHLLQLTVWGIKQL
gp160
562-576
human
VWGIKQLQARVLAVERYLKD
gp160
570-589
human
YLRDQQLLGIWG
gp160
586-597
human, chimpanzee
LGIWGCSGKLIC
gp160
593-604
human
DR4(Dw10)
DR
DR
DR
GIWGCSGKLI
gp160
594-603
human
CSGKLICTTAVP
gp160
598-609
human
CTTAVPWNASWS
gp160
604-615
human
PWNASWSN
gp160
609-616
human
WSNKSLEDIWDNMTWC
gp160
614-629
human
EIDNYTNTIYTLLEEC
gp160
634-649
human
EESQNQQEKNEQELL
gp160
647-661
human
QNQQEKNEQELLE
gp160
650-662
human
ASLWNWFNITNWLWY
gp160
667-681
human
IKLFIMIVGGLVGLR
gp160
682-696
human
GIEEEGGERDRDR
gp160
732-744
human
WLNATAIAVTEGTDRC
gp160
814-829
human
YVAEGTDRVIEVVQGACR
gp160
821-838
human
DRVIEVVQGAYRAIR
gp160
827-841
human
AIRHIPRRIRQGLER
gp160
839-853
human, mouse
HIPRRIRQGLERILL
gp160
842-856
human
H-2k, H-2b, H-2s
Published Env-specific Immunodominant CD4 T epitopes
based on HXB2 sequence
Epitope
Protein
HXB2
Species HLA
Location
MGARASVLSGGELDRWEK
p17
1-18
human
SGGELDRWEKIRLRPGGK
p17
9-26
human
EKIRLRPGGKKKYKLKHI
p17
17-34
human
LRPGGKKKYKLKHIV
p17
21-35
human
RPGGKKKY?
p17
22-29
human
KHIVWASRELERFAV
p17
32-46
human
HIVWASRELERFAVN?
p17
33-47
human
DR13.02
ASRELERFAVNPGLL
p17
37-51
human
DRB*0101,DRB1*0401,
DRB1*0405,DRB1*0701,
DRB1*1302, DRB1*1501
RELERFAVN
p17
39-47
human
DRB1*1302
LERFAVNPGLL
p17
41-51
human
DRB1*1302
ERFAVNPGLL
p17
42-51
human
DRB3*0202, DRB3*0301
ERFAVNPGLLETSEGCR
p17
42-58
human
DRB1*0101,DRB1*0405,
RB1*1101, DRB1*1302
TGSEELRSLNTVALY
p17
70-86
human
DRB1*0101, DRB1*0401,
DRB1*0405, DRB1*0701,
DRB1*1302, DRB5*0101
SLYNTVATLYCVHQRIEV
p17
77-94
human
EIKDTKEALDKIEEE
p17
93-107
human
AAADTGHSSQVSQNY
p17
118-132
human
PIVQNIQGQ
p24
1-9
human
DRβ1*0101
PIVQNLQGQMV
p24
1-11
human
DR1
PIVQNIQGQMVHQAI
p24
1-15
human
QGQMVHQAISPRTLN
p24
7-21
human
DR supermotif
QGQMVHQAISPRTLN
p24
7-21
mouse
I-Ab and HLA-DR
QMVHQAISPRTLNAWVKV
p24
9-26
human
VHQAISPRTLNAWVKC
p24
11-26
human
NAWVKVVEEKAFSPEC
p24
21-36
human
WVKVVEEKAFSPEVIPMF
p24
23-40
human
EEKAFSPEV
p24
28-36
human
DRβ1*0101
EEKAFSPEVIP
p24
28-38
human
DQ5
AFSPEVIPMFSALSEC
p24
31-46
human
A*0201
AFSPEVIPMFSALSEGA
p24
31-47
human
PEVIPMFSALSEGATP
p24
34-49
human
DR1
EVIPMFSALS
p24
35-44
human
DRβ1*0101
EVIPMFSALS
p24
35-44
human
DR4
SALSEGATPQDLNTMC
p24
41-56
human
TPQDLNTMLNTVGGH
p24
48-62
human
DLNTMLNTYGGHQAAC
p24
51-66
human
LKETINEEAAEWDRVHPVHA
p24
69-88
human
ETINEEAAEWDRVHPC
p24
71-86
human
ETINEEAAEWDRVHPVHA
p24
71-88
ETINEEAAEWDRVHPVHA
p24
71-88
human
EAAEWDRVHP
p24
76-85
human
EAAEWDRVHPVHAGP
p24
76-90
human
EWDRVHPVHA
p24
79-88
human
DR
DRβ1*0101
DRβ1*0101
DRβ1*0101
DRVHPVHAGPIAPGQ
p24
81-95
macaque
VHAGPIAPG
p24
86-94
human
HAGPIAPGQMREPRG
p24
87-101
mouse
MREPRGSD
p24
96-103
human
MREPRGSKIAGTTST
p24
96-110
human
EPRGSDIAGT
p24
98-107
human
DQ7
PRGSDIAGTTSTLQEQIGWM
p24
99-118
human
DR4
GSDIAGTTSTLQEQI
p24
101-115
macaque
GSDIAGTTSTLQEQIC
p24
101-116
human
STLQEQIGWMTNNPPIPVGE
p24
109-128
human
TNNPPIPBGEIYKRW
p24
119-133
human
NPPIPVGEIYKRWIIC
p24
121-136
human
NPPIPVGEIYKRWILGLNK
p24
121-140
mouse
H2
NPPIPVGEIYKRWIILGLNK
p24
121-140
mouse
H-2
DQ7
DRB1*1301
NPPIPVGEIYKRWIILGLNK
p24
121-140
mouse
H-2d
GEIYKRWIILGLNKI
p24
127-141
human
DR supermotif
EIYKRWIILG
p24
128-137
human
DRB1*1301, DRB1*1302
IYKRWIILGLNKIVRMYSPT
p24
129-148
human
KRWIILGLNKIVRMY
p24
131-145
macaque
KRWIILGLNKIVRMY
p24
131-145
human
DR supermotif
human
DRB1*0101, DRB1*1101,
DRB1*1302, DRB1*1501,
DRB5*0101
WIILGLNKIVRM
p24
133-144
WIILGLNKIVRMYSPTSI
p24
133-150
human
DRB1*0101, DRB1*0401,
DRB1*0405, DRB1*0701,
DRB1*1101, DRB1*1302,
DRB1*1501, DRB5*0101
ILGLNKIVRMY
p24
135-145
human
DRB1*0401, DRB1*1302,
DRB1*1501
ILGLNKIVRMYSPTSILDIR
p24
135-154
human
NKIVRMYSPT
p24
139-148
macaque
NKIVRMYSPTSILDIRQGP
p24
139-157
human
DR4
KIVRMYSPT
p24
140-148
human
DRβ1*0101
IVRMYSPTSILDIRQC
p24 141-156
human
IVRMYSPTSILDIRQGPK
p24 141-158
human
SPTSILDIRQGPKEP
p24 146-160
human
SILDIRQGPKEPFRDYVDRF
p24 149-168
human
ILDIRQGPKEPFRDYVDRFY
p24 150-169
human
LDIRQGPKEPFRDYVC
p24 151-166
human
GPKEPFRDYVDRFYK
p24 156-170
human
GPKEPFRDYVDRFYKTLR
p24 156-173
human
QPKEPFRDYVDRFYKTLRA
p24 156-174
human
PKEPFRDYV
p24 157-165
human
DQ5
FRDYVDRFYKTLRAEQASQD
p24 161-180
mouse
H-2, H-2
DYVDRFYKTLRAE
p24 163-175
human
DR0101
DR4
DYVDRFYKTLRAEQA
YVDRFYKTLRAEQASQEV
RFYKTLRAEQAS
FYKTLRAEQASQ
p24
p24
p24
p24
163-177
164-181
167-178
168-179
human
DRB1*1302
human
DRB1*0101,
DRB1*0401,
DRB1*0405,
DRB1*0701, DRB1*1101,
DRB1*1302,
DRB1*1501,
DRB5*0101
human
DRB1*0101,
DRB1*0401,
DRB1*0405,
DRB1*0701, DRB1*1101,
DRB1*1501,
DRB5*0101
human
DRB1*0101,
DRB1*0401, DRB1*1101,
DRB5*0101
FYKTLRAEQASQE
p24
168-180
human
DRB1*0101,
DRB1*0401,
DRB1*0405,
DRB1*1101,
DRB1*1501,
DRB5*0101
YKTLRAEQA
p24
169-177
human
DRB1*0101
YKTLRAEQAS
p24
169-178
macaque
VKNWMTETLLVQNANC
p24
181-198
human
MTETLLVQNANPDCKTIL
p24
185-202
human
GNFRNQRKIVKCFNCGKEGH
p2p7p1p6 18-37
human
FNCGKEGHTARNCRA
p2p7p1p6 30-44
human
HIAKNCRAPRKKGCWK
p2p7p1p6 37-52
human
DR15/51
KEGHQMKDCTERQAN
p2p7p1p6
55-69
human
MKDCTERQANFLGKI
p2p7p1p6
60-74
human
RQANFLGKIWPSHKGR
p2p7p1p6
66-81
human
GKIWPSHKGRPGNFLQSR
p2p7p1p6
72-89
human
PSYKGRPG
p2p7p1p6
76-83
human
GNFLQSRPEPTAPPA
p2p7p1p6
83-97
mouse
TAPPEESFRFGEETTTPSQK
p2p7p1p6
93-112
human
ESFRSGVETTTPPQK
p2p7p1p6
98-112
human
REETTTPS
p2p7p1p6
103110
human
QKQEPIDKELYPLASLR
p2p7p1p6
111127
human
KELYPLASLRSLFGNDPSSQ
p2p7p1p6
118137
human
DRB1*0101, DR01*0401,
DRB1*0405, DRB1*1101,
DRB1*1302,
DRB1*1501,
DRB5*0101
H-2
T-cell-immunome-discovery
flow chart.
• Bioinformatics tools for
selecting protein subsets (by
searching for motifs
corresponding to secretion
signals or transmembrane
domains) combined with
molecular tools, such as
microarrays, allow the selection
of a subset of genes from
genomic sequences for further
in silico screening.
• Epitope-mapping tools allow
the selection of the ensemble
of epitopes within these proteins
that could interact with the host
cellular
immune system.
• Confirmation of the
immunogenicity of these
epitopes can be obtained in vitro
(using HLA binding assays
and/or T-cell assays) or in vivo,
in HLA transgenic mice.
Phage display or peptide display may
offer another microarray strategy in
defining the functional epitopes
for vaccine research
Polyvalent M13-g8p phage display
M13 phage
Peptide M13 g8p fusion
Viral peptides (30-50 mer)
Affinity selection of the HIV/HCV
peptide display library using convalescent
human sera samples
Magnetic beads
protein G
Magnetic beads were
coated with protein G
(Dynabeads M280
tosylactivated, Dynal)
Human serum:
- non-infected and infected
- Acute or chronic Infected
- Protected or non-protected
- HARRT/ART treated or
non-treated
- mono-infected or coinfected
Phage display
Phage array displaying immunodominant epitopes
Specific
Non-specific
Binding
Specific
Panning
Specific
Elution
E.Coli infection
Differential dilution
plating
Plaque characterization: sequencing and mapping
Data assessing Immunodominant epitopes identification and phage stocking
Replica
Phage array for T cells research and vaccine development
Thank you for your attention