Transcript P - World Congress on Virology

Transcription Profiling of CD4+ T Cells in
Rhesus Macaques that Infected with Simian
-Human Immunodeficiency Virus and Rechallenged with SIVmac251
5th World Congress on Virology
Dec 07-09, 2015 Atlanta
Hye Kyung Chung Ph.D
Advanced BioScience Laboratories, Inc
Viral Load
SHIV infection Patterns
Post-Infection
2
THANK YOU
Advance BioScience Laboratories
National Cancer Institute, NIH
John Brady
Ranajit Pal
Lindsay Gregory
Michael Lee
Allison Younkins
Ahae Woo
Gayatri Patel
Cindy Masision
Mike Rodonovichi
Moffitt Cancer Center , University of South Florida
EunMi Lee
Jessica Livesay
Anthony Cristillo
Gerald Kovacs
Jae K. Lee
Annamalai Muthiah
Soo-Young Cheon
Animal Facility
Debora Weiss
Jim Treece
•
Support: Partial Support by NIH/NIAID contract
N01-AI-15430 to Advanced BioScience Laboratories
Purpose
•
We hypothesize that there are differences among
animals in their ability to control virus infection by
mounting appropriate immune responses.
•
We believe that the molecular basis for these
differences can be determined by analyzing gene
expression profiles.
•
To test this hypothesis, we are comparing global
gene expression patterns in rhesus macaques that
control –or do not control- persistent virus
replication.
In vaccine research, we need to have better markers to
predict whether a vaccine will be protective.
Using the power of modern genetic analysis such as
microarray, we can establish a “signature” or gene
expression profile that defines the protective immune
response.
This technology has been used successfully to establish
gene expression signatures, ALL-AML
class Prediction in tumors.
Science, 1999
5
Contents
Part 1 Innate Host Immune Responses: Natural Control of
SHIV Replication
Part 2 Innate and Adaptive Antiviral Immune Responses
Biomarkers of Innate and Adaptive Immunity in CD4+ T Cells
6
Part 1 Innate Immunity
•
An ideal vaccine stimulates four components of the immune system. 1) Elicit
neutralizing antibody at high titer. 2) Stimulate a cellular (T-Cell) immune
response, especially cytotoxic T-cells. 3) Stimulate mucosal immunity. 4)
Provoke the innate immune system.
•
The rhesus macaque has served as an animal model system for vaccine
development and also to study pathogenesis of several human infectious
diseases.
•
Microarray technology has been extensively used to analyze a global gene
expression pattern in cancer and other diseases to monitor the molecular
mechanism of pathogenesis.
•
Knowledge of natural host defense mechanisms may lead to their exploitation
for therapeutic or prophylactic purposes.
7
SHIVsf162P3 RNA levels in Plasma in
Virus Infected Rhesus Macaques
Viral RNA Copies/ml Plasma
108
713L
714L
717L
718L
719L
720L
68M
69M
107
106
108
107
106
105
105
104
104
103
103
0
20
40
60
80
100
Weeks Post Challenge
120
140
0
10
20
30
40
Days Post Challenge
Chung, et al., Viral Immuno. 2008
50
Gene Expression Profiles in PBMC from Naïve
vs SHIVsf162P3 infected Rhesus Macaques
Condition
Animal
Time
Controlled
714L, 718L
Day 21
High
714L, 718L
>Year 2
Low
713L, 720L
Day 21
High
713L, 720L
>Year 2
Non-controlled
Naïve
Viral load
High
Pool of 8 uninfected monkeys
9
Clustering Heatmap of Differentially
Expressed Genes
Day 21
Year 2
B. Year
A. Day
A2N713Y
AN720Y
A2C714Y
200648_s_at
204351_at
204860_s_at
203922_s_at
201670_s_at
205237_at
206715_at
210029_at
212335_at
209970_x_at
204924_at
207857_at
204588_s_at
202609_at
206343_s_at
215223_s_at
211367_s_at
212820_at
216841_s_at
212268_at
212224_at
209499_x_at
206710_s_at
211404_s_at
212192_at
203535_at
203041_s_at
203574_at
209230_s_at
212636_at
218728_s_at
201647_s_at
215001_s_at
202241_at
222156_x_at
202388_at
204533_at
217502_at
202411_at
214453_s_at
211368_s_at
205552_s_at
205660_at
206420_at
209969_s_at
202269_x_at
205483_s_at
201739_at
205842_s_at
201616_s_at
213516_at
214894_x_at
215594_at
221225_at
204838_s_at
212289_at
217340_at
207979_s_at
213109_at
AC718Y
AN720D
A2N713D
AC718D
A2C714D
207979_s_at
204838_s_at
214894_x_at
201616_s_at
215594_at
217340_at
212289_at
221225_at
213109_at
213516_at
205660_at
211368_s_at
205483_s_at
205552_s_at
209969_s_at
202269_x_at
214453_s_at
202411_at
204533_at
217502_at
212636_at
202241_at
218728_s_at
209970_x_at
206715_at
211367_s_at
210029_at
201739_at
204924_at
222156_x_at
205842_s_at
202388_at
215001_s_at
203574_at
204351_at
206420_at
203041_s_at
201647_s_at
202609_at
206710_s_at
204588_s_at
212192_at
212224_at
212820_at
212335_at
212268_at
201670_s_at
200648_s_at
204860_s_at
216841_s_at
206343_s_at
209499_x_at
209230_s_at
205237_at
211404_s_at
203535_at
203922_s_at
207857_at
215223_s_at
Chung, et al., Viral Immunol. 2008
Heatmap of Differentially Expressed
Genes at Day 21 and Year 2
C. Day+Year
A2N713Y
AN720Y
AN720D
A2N713D
A2C714Y
AC718Y
AC718D
A2C714D
209970_x_at
204924_at
211367_s_at
201739_at
210029_at
206715_at
204533_at
217502_at
209969_s_at
202269_x_at
202411_at
214453_s_at
205660_at
211368_s_at
205552_s_at
205483_s_at
218728_s_at
215001_s_at
202388_at
202241_at
203574_at
212636_at
222156_x_at
206420_at
205842_s_at
206710_s_at
202609_at
204588_s_at
203041_s_at
201647_s_at
204860_s_at
216841_s_at
201670_s_at
200648_s_at
212224_at
212268_at
204351_at
212820_at
212335_at
206343_s_at
203922_s_at
207857_at
215223_s_at
209499_x_at
212192_at
209230_s_at
205237_at
211404_s_at
203535_at
204838_s_at
214894_x_at
207979_s_at
201616_s_at
215594_at
221225_at
213516_at
212289_at
217340_at
213109_at
Chung, et al., Viral Immuno. 2008
Part 1 Summary
We were examined differences between protective and non-protective host
response by measuring global cellular gene expression profiles in SHIVinfected macaques.
Comparing gene expression profiles in PBMC from animals that exhibit
different levels of virus control showed interesting differences in gene
expression pattern.
We identified 59 genes. Activation of host defense-related genes such as
interference-inducible genes, apoptosis genes, and inflammation-related
genes are up-regulated in the non-controllers.
The results suggested that these genes might contribute to a favorable
microenvironment for virus replication in vivo.
It could help identify early parameters that predict the efficiency of protective
immune responses.
12
Part 2 Innate and Adaptive Immune
Responses
0
5.2 y
Group 1 N=3
SHIVsf162P3
0
SIV mac 251
Day 3
Day10
Day 28
Day 160
Day 3
Day 10
Day 28
Day 60
2.7 y
Group 2 N=3
SHIVBaL
SIV mac251
13
Characteristics of Rhesus Macaques Used for
Global Gene Expression Analysis
Animal* Weight
714L
717L
718L
184M
185M
186M
8.9
10.8
11.2
8.7
8.7
8.4
Sex
Male
Male
Male
Male
Male
Male
Prior to SIV mac251
Challenge
SHIVsf162P3
SHIVsf162P3
SHIVsf162P3
SHIV BaL
SHIV BaL
SHIV BaL
Re-Challenge
Virus
SIVmac251
SIVmac251
SIVmac251
SIVmac251
SIVmac251
SIVmac251
Challenge
Route
Rectal
Rectal
Rectal
Rectal
Rectal
Rectal
14
MHC Class 1 Genotyping
Animal ID
714L
717L
718L
184M
185M
186M
Mamu-A*01a
Negative
Negative
Negative
Negative
Negative
Positive
Mamu-B*08b
Negative
Positive
Negative
Negative
Negative
Negative
Mamu-B*17b
Negative
Negative
Negative
Negative
Negative
Negative
15
TRIM 5 alpha Genotyping
G/T
Animal
T307 P327 Q332
Splice Site
ID
P
T
R
Mutation
714L
T/P P/T Q
G/G
717L
T/P P/T Q
G/G
718L
G/T
T
P
Q
184M
P
P
Q
G/G
P
P
Q
185M
G/T
186M
T/P P
Q
G/G
Moderate
Resistance
333
ACGTTT C1002A
334P
A/S/
Genotypesa
/Q/R
T
TF339-340 P341Q
A/S P
TRIM TFP/TRIM Q
TF/∆∆
P/Q
A/S P
TRIM TFP/TRIM Q
TF/∆∆
P/Q
TRIM TFP/TRIM CYPA
A
P
TF
P
A/S P
TRIM TFP/TRIM Q
TF/∆∆
P/Q
S
P
TRIM Q/TRIM CYPA
∆∆
Q
A
P
TRIM TFP/TRIM TFP
TF
P
Most Resistance
16
Virological Outcome in Plasma/PBMC at Preand 160 days Post-Rechallenge
Animal
714L
717L
718L
184M
185M
186M
Prior to SIV mac251 Re-challenge
Viral RNA
SIV proviral DNA copy Load/ml of
number / 106 PBMC
Plasma
<10
<50
<10
<50
3.37E+01
<50
1.53E+02
<50
5.39E+02
<50
3.29E+02
<50
160 days Post Challenge
Viral RNA
SIV proviral DNA copy Load/ml of
number / 106 PBMC
Plasma
1.03E+02
509
7.87E+01
<50
1.58E+02
64,192
3.52E+02
346
4.85E+03
61,638
2.45E+02
23,635
17
Viral RNA Levels in Plasma in SIVmac251
Infected Macaques
106
108
Viral RNA Load (Copies/ml)
Viral RNA Load (Copies/ml)
184M
185M
186M
107
106
105
104
103
714L
717L
718L
105
104
103
102
102
101
101
0
20
40
60
80
100
Days Post Infection
120
140
160
180
0
20
40
60
80
100
120
140
160
180
200
Days Post Infection
18
Chung, et al., J Med Primatol. 2015
Heatmap for Differentially Expressed Genes between
Protected and Un-Protected Rhesus Macaque
19
Heatmap for Differentially Expressed Genes with
Human and Rhesus Macaque Chips
20
Network Diagram Derived from Eight Common
Genes of Human Chip Microarray Data
21
Network Diagram Derived from Eight Common Genes of SIV mac251
Re-Challenge Study using Monkey Chip Microarray Data
22
Eight Protective Biomarker
Human chip
Monkey chip
Exponential fold changes
Exponential fold changes
Gene Symbol
Probe Set ID
MX1
202086_at
2.61
2.23
IFI27
202411_at
6.83
2.23
S100A9
203535_at
5.33
1.18
TYROBP
204122_at
2.34
1.30
LMO2
204249_s_at
1.71
1.32
MX2
204994_at
2.10
1.77
FCN1
205237_at
4.35
1.52
JAK2
205842_s_at
2.35
1.74
23
Part 2 Summary
Our finding suggested that SHIV a model of attenuated viral vaccineinduced protective immune response from pathogenic SIVmac 251
infection.
Down regulation of certain interferon responses and tyrosine kinase
pathway may indicate protective vaccine responses.
The eight common genes MX1, MX2, IFI27, JAK2, LMO2, TYROBP, FCN1,
and S100A9 with P values <0.01 were identified as a potential protective
biomarker, and all genes were down-regulated in the protected macaques.
24
Conclusions
Carefully designed global gene expression profiling
could help identify early parameters that predict the
protective immune responses in HIV vaccine evaluation
in NHP model.
Despite the fact that we were able to identify statistically
significant differences in expression levels between
protected and unprotected animals these observations
were warrant validation using larger study groups.
25
THANK YOU
Advance BioScience Laboratories
National Cancer Institute, NIH
John Brady
Ranajit Pal
Lindsay Gregory
Michael Lee
Allison Younkins
Ahae Woo
Gayatri Patel
Cindy Masision
Mike Rodonovichi
Moffitt Cancer Center , University of South Florida
EunMi Lee
Jessica Livesay
Anthony Cristillo
Gerald Kovacs
Jae K. Lee
Annamalai Muthiah
Soo-Young Cheon
Animal Facility
Debora Weiss
Jim Treece
•
Support: Partial Support by NIH/NIAID contract
N01-AI-15430 to Advanced BioScience Laboratories