Embed Size (px)
Citation preview
Plasma cytokines and chemokines detection
KSHV PCR-detection in tissues compartments of KS patients
Common KS lesions at ORCI – Tanzania (IRB approval and patient consent was obtained)
• Kaposi’s sarcoma (KS) is a multifocal angio-proliferative tumor with mucocutaneous and visceral involvement. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent.
• HIV-1/AIDS associated/epidemic (EpKS) and the African endemic (EnKS) are common in sub-Saharan Africa.
• KSHV infection is ~60% in US HIV-1+ individuals and other high-risk groups • There is no vaccine or curative treatment for KS.
• The high KS incidence in HIV-1 positive individuals and transplant recipients on immunosuppressive drugs, points to immune dysfunction as underlying KS development.
• The role of HIV-1 in KS tumorigenesis is unclear, and mechanisms driving EnKSare poorly understood.
Study subjects (N=131)
To compare humoral immune responses and tumor gene expression profiles between EpKS and EnKS patients in order to reveal unique pathways of viral neoplasia and better define the role of HIV-1 co-infection.
Background Antibody/Cytokines responses
Conclusions
Goal
Acknowledgments
• KSHV PCR using tumor biopsy is the most reliable method for detection of KSHV DNA. Variable detection in other compartments.
• Similar anti-KSHV antibody responses between EnKS and EpKS despite HIV-1 co-infection in EpKS patients.
• Cytokines dyregulation is similar in EnKS and EpKS despite HIV-1 co-infection.• EnKS is similar to EpKS at the transcriptomic level.• Treatment of HIV-1 infection with ART does not alter the KS transcriptome profile.• KSHV expression (load) had substantial impact on the cellular transcriptome
profile. • KS is primarily a KSHV disease and HIV-1 exacerbates KSHV pathogenesis.
Future directions• Cell-mediated responses to specific viral peptides are being assessed.• Longitudinal immune responses are being quantified in correlation with KS-
specific and HIV-1 treatment outcomes.
• All study participants and clinical team members.• Contributing members of Wood/West lab.• This project is supported by NIH/NCI (RO1 CA75903 and U54 CA190155), FIC (D43
TW010354) and NIGMS (P30 GM103509).
Non-cancer controls KS Patients P-values
VariablesHIV-1-KSHV-N=20
HIV-1-KSHV+ N=27
HIV-1+KSHV-N=11
HIV-1+KSHV+ N=22
EnKS N=17
EpKSN=34
HIV-1-KSHV+ vs
EnKS
HIV-1+KSHV+ vs
EpKS
EnKS vs EpKS
Median age (Range) 35 (21-52) 28 (21-60) 40 (24-50) 38 (29-68) 51 (24-87) 37 (21-60) 0.0009 0.5070 0.0490Male (%) 7 (35) 13 (48.1) 5 (45.5) 11 (50) 16 (94.1) 19 (55.9) 0.0020 0.6692 0.0060
ART treatment (%) 9 (81.8) 13 (59.1) 30 (88.2) 0.0124Median ART
duration-months (Range)
4 (0.25-144) 9 (0.1-48) 5 (0.1-
120) 0.7690
Median CD4:CD8 ratio 1.58 1.56 0.47 0.20 1.27 0.18 0.4357 0.1510 < 0.0001
Individuals with lasma HIV detection
(Range in copies/mL)
3 (<50-1.7x106)
10 (<50-1.4x107)
14 (<50-1.7x106) 0.7542
Median KS duration-months (Range) 12 (3-108) 5 (5-96) 0.0030
KS Tumor Plasma PBMC N
EnKS
+ - + 4
+ + - 4
+ - - 6
+ + + 2
EpKS
+ - + 7
+ + - 3
+ - - 5
+ + + 15
KS lesion is most reliable for KSHV DNA detection compared to other compartments.
Similar cytokine dysregulation in EnKS and EpKS patients despite HIV-1 infection in EpKS
KS lesion Plasma PBMC0
20
40
60
80
100
EnKSEpKS
* *P=0.019
KSH
V de
tect
ion
(%)
+ sampleImmunofluorescence assay
KSHV AgBC3 cells
1˚ α-KSHV Ab from plasma
2˚ Mu α-human Ig
✳︎3˚ Dnk α-mouse 488nM
- sample
Similar anti-KSHV Ab titers in EnKS and EpKS despite HIV-1 and low CD4:CD8 ratio in EpKS patients.
HIV-1-KSHV- HIV-1-KSHV+ HIV-1+KSHV- HIV-1+KSHV+ EnKS EpKS1
10
100
1000
10000
100000P<0.0001
P<0.0001
Plasma HIV-1 viremic
Anti-
KSH
V an
tibod
y tit
er(R
ecip
roca
l end
poin
t pla
sma
dilu
tion)
KSHV neutralization antibody (nAb) assayKS patient plasma dilution
eGFP if KSHV infected
Flow cytometryrKSHV.219 from Vero.219 cells induced with RTA
nAb+ nAb-
High prevalence and titer of nAb in both EnKS and EpKS patients compared to cognate controls.
HIV-1-KSHV- HIV-1-KSHV+ HIV-1+KSHV- HIV-1+KSHV+ EnKS EpKS1
10
100
1000
10000 P=0.0006P<0.0001
Plasma HIV-1 viremic
nAb
reci
proc
al IC
50
≤ -15-10-5-2±12510
≥ 20
Fold
02003005007001000150020005000
KSHVreads/M
p211
p224
p235
p236
p239
p240
p132
p122
p183
p123p229
p222
p228
p211p224p235
p236
p239p240
p132
p122
p183
p123
p229
p222
p228
p335 p334
p326
p325p323
p338
-2
0
2
4
6
8
10
12
14
-200 -150 -100 -50 0 50 100 150 200
KSHV
, log
2(1+
RNA
load
)
Principal component 1 (47%)
ControlKS endemicKS epidemic, ARTKS epidemic, untreated
r = 0.788p = 4x10-9
p211p224
p235
p236
p239
p240
p132
p122
p183
p123
p229
p222
p228
p335
p334
p326
p325
p323
p338
p211p224
p235
p236
p239
p240
p132
p122
p183
p123
p229
p222
p228
p335
p334
p326
p325
p323
p338
-100
-80
-60
-40
-20
0
20
40
60
80
100
-200 -150 -100 -50 0 50 100 150 200
Prin
cipa
l com
pone
nt 2
(18%
)
Principal component 1 (47%)
ControlKS endemicKS epidemic, ARTKS epidemic, untreated
RNA extraction
with DNAse
digestion
Sample
fragmentation
bar-coding and
Illumina HiSeq
Bioinformatics analyses
RNA quality
assessment
Cellular genes; Endemic vs Epidemic & ART treated vs untreated
KSHV genes
RNA seq. KS/control
KS was confirmed by PCR amplification of KSHV DNA from tumor biopsies.
EnKS tumor gene expression is indistinguishable from EpKS.
1Nebraska Center for Virology, 2School of Biological Sciences, 3Department of biochemistry University of Nebraska-Lincoln, Lincoln, NE, USA; 4Ocean Road Cancer Institute, Dar es Salaam, Tanzania; 5Dermatology and Venereology section, University Teaching Hospitals, University of Zambia School of Medicine, Lusaka, Zambia; 6Wistar Institute, Philadelphia, USA; 7Department of Epidemiology,
7College of Public Health, University of Nebraska Medical Center, Omaha, NE, USA
Salum J. Lidenge1,2,4, For Yue Tso1,2, Andrew V. Kossenkov6, Owen Ngalamika5, John R. Ngowi4, Yasaman Mortazavi1,2, Eun Hee Kwon1, Danielle M. Shea1, Veenu Minhas7, Julius Mwaiselage4, Paul M. Lieberman6, Charles Wood1,2, and John T. West1,3
Primary Role of KSHV in Pathogenesis of Endemic and Epidemic Kaposi’s Sarcoma0270
KS/ KS typecontrol treatement
fold KSHV load136 ORF7570 K1258 K729 ORF7226 ORF7323 ORF7118 K213 ORF5012 ORF59
10.0 K89.8 K59.8 ORF577.3 ORF26.9 ORF115.2 ORF45.1 vIRF-44.8 ORF374.4 ORF614.3 ORF104.2 ORF483.7 ORF353.5 ORF603.5 ORF67A3.4 ORF453.1 ORF473.1 ORF463.0 K43.0 ORF623.0 ORF62.8 K152.7 ORF682.6 vIRF-32.5 K142.5 ORF362.5 ORF442.5 K32.4 ORF392.4 ORF652.4 ORF17.52.3 ORF542.3 ORF432.2 vIRF-22.2 ORF522.2 ORF742.2 ORF172.2 K62.1 ORF402.1 ORF332.0 K4.22.0 ORF25
1.99 ORF631.95 ORF291.92 ORF581.84 ORF561.75 ORF221.75 ORF271.75 ORF701.73 ORF671.72 ORF311.70 K4.11.69 vIRF-11.67 ORF261.65 ORF341.64 ORF421.54 ORF661.50 ORF241.49 ORF301.47 ORF551.42 ORF91.42 ORF211.39 ORF641.37 ORF691.36 ORF531.36 ORF191.32 K8.11.30 ORF181.30 ORF231.24 ORF491.20 ORF321.16 ORF20
Endemic Epidemicno HIV under ART no HIV treatment
• Latency and immunomodulatory KSHV genes are highly expressed in KS tumors.
• KS tumors overexpress IFN-associated chemokines.
• Altered glucose and lipid metabolism in KS.
Plasma HIV-1 viremic
Regulators & downstream target genes
Significantly changed cellular genes in KS lesions (IPA)Metabolic disorders in the KS lesions
