Functional Microbial GenomicsHIV

1
Functional Microbial GenomicsHIV

• Shainn-Wei Wang, Ph.D.
• NCKU, College of Medicine
• Institute of Molecular Medicine

2
HIV exhibits tremendous genetic diversity
Garber D. A., et al., Lancet Infec. Dis., 2004
3
HIV Data Base

• http//www.hiv.lanl.gov/content/index

4
HIV/HCV Database
http//lasp.cpqgm.fiocruz.br/LaspIngles/ferramenta
s.html
5
(No Transcript)
6
(No Transcript)
7
http//www.flu.lanl.gov/
8
(No Transcript)
9
(No Transcript)
10
(No Transcript)
11
Human Immunodeficiency Virus (HIV)
12
(No Transcript)
13
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)
Latent infection
Acute infection
14
Infection and reservoir
15
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

16
Mock control
PBMC Target cells
Infection signature gene sets
Non-Infection gene sets
comparison
Change in disease or functional Status
17
The Journal of Infectious Diseases    2004189572
-582
18
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

19
(No Transcript)
20
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

21
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.

22
What they found in the array

• HIV-1 Tat induces expression of
  interferon-inducible genes
• HIV-1 Tat mediates chemokine upregulation in iDC

23
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 ß-actin mRNA was not affected by HIV-1
  or adeno-Tat, confirming the microarray analysis.

24
Rt-PCR gene expression analysis in iDC obtained
from donors
A, B, C,D donors
25
MCP-2 expression and SIV infection in axillary
lymph nodes
a, SIV Nefexpressing 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. ce, 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 ce shows a single SIV-infected dendritic cell
(indicated by arrow) expressing all three markers
(original magnification a,b, 200, cf, 400).
26
Chemotaxis of Monocytes and activated T cells
27
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
28
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-? (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-? 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

29

• 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-a, IL-1,
  IL-6, IL-10, 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

30
Genomic Database for HIV Infection
31
(No Transcript)
32
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

33
Hepatocyte-growth factor-Regulated tyrosine
kinase Substrate
The Protein Network of HIV Budding, Cell, Vol.
114, 701713, September 19, 2003
34
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.

35
(No Transcript)
36
Tandem Affinity Purification Method
Guillaume Rigaut, 1999
TEV cleavage site
calmodulin-binding peptide
protein A
TAP tag
37
Tandem Affinity Purification Method
Protein composition of TAP-purified U1 snRNP.
Guillaume Rigaut, 1999
38
Tandem Affinity Purification Method
Guillaume Rigaut, 1999
39
Tandem Affinity Purification Method
Protein composition of TAP-purified U1 snRNP.
Guillaume Rigaut, 1999
40
Tandem Affinity Purification Method
Protein composition of TAP-purified U1 snRNP.
Guillaume Rigaut, 1999
41
Protein identification and functional analysis
MALDI-TOF
Protein samples from GE
- Online Data mining - Functional assay
42
Nature. 2002 Jan 10415(6868)141-7.
43
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).
44
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.
45
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.
46
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

47
(No Transcript)
48
CTL activity
HIV encoded proteins
CD4 or function
MHC
HIV viral load

• Viral genetic diversity
• - individuals infected with different viral
  clades show different response patterns

• MHC genetic variability in human populations

Disease Protection or Disease progression
Caucasian Africans Asians Hispanics South
American Indians

• these populations differ in their HLA allele
• frequencies
• the HIV-specific responses detected in
• these ethnicities can also differ
• significantly.

49
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

50
Nat Med. 2005 Dec11(12)1290-2. Epub 2005 Nov 20
51
The epitopes tested are described in the Los
Alamos database (www.hiv.lanl.gov contentimmunolog
ytablesctlsummary.html) in the context of HIV
infection and are listed in Table 2
52
(No Transcript)
53
(No Transcript)
54
(No Transcript)
55
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).
56
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.

57
In searching HIV and HCV antigens involved in
protective immunity.
58
Published 22 Env-specific Immunodominant CD4 T
epitopes based on HCV 77
59
Published 22 Core-specific Immunodominant CD4 T
epitopes based on HCV 77 sequence
60
Published 22 Env-specific Immunodominant CD4 T
epitopes based on HCV 77 sequence
61
Published Env-specific Immunodominant CD4 T
epitopes based on HXB2 sequence
62
(No Transcript)
63
(No Transcript)
64
(No Transcript)
65
(No Transcript)
66
Published Env-specific Immunodominant CD4 T
epitopes based on HXB2 sequence
67
(No Transcript)
68
(No Transcript)
69
(No Transcript)
70
(No Transcript)
71
(No Transcript)
72
(No Transcript)
73
(No Transcript)
74
(No Transcript)
75
(No Transcript)
76
(No Transcript)
77
(No Transcript)
78

• 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.

79
(No Transcript)
80
(No Transcript)
81
(No Transcript)
82
Phage display or peptide display may offer
another microarray strategy in defining the
functional epitopes for vaccine research
83
Polyvalent M13-g8p phage display
M13 phage
Peptide M13 g8p fusion
Viral peptides (30-50 mer)
84
Affinity selection of the HIV/HCV peptide display
library using convalescent human sera samples
Magnetic beads
protein G

• Human serum
• non-infected and infected
• Acute or chronic Infected
• Protected or non-protected
• HARRT/ART treated or
• non-treated
• mono-infected or coinfected

Magnetic beads were coated with protein
G (Dynabeads M280 tosylactivated, Dynal)
Phage display
85
Phage array displaying immunodominant epitopes
Specific
Binding
Non-specific
Specific
Panning
Specific
Elution
E.Coli infection Differential dilution
plating
Plaque characterization sequencing and mapping
Data assessing
Immunodominant epitopes identification and phage
stocking
Phage array for T cells research and vaccine
development
Replica
86
Thank you for your attention