Transcript Innate Immune Responses

Rapid and systemic
innate immune responses
to an adenoviral HIV vaccine
Erica Andersen-Nissen
McElrath Lab
Fred Hutchinson Cancer Research Center
Developing an HIV vaccine
• Very challenging task!
• Two potential scenarios for an HIV vaccine:
-slow clinical disease
progression
-reduce transmission
DH Barouch
Nature 455,
613-619 (2008)
• Regardless of the type of HIV vaccine developed,
we need to better understand how to induce a
protective adaptive response
– > innate immune response
Arms of the Immune System
When a microbe invades, the immune system:
• Recognizes it
• Mobilizes and eliminates it
• Remembers it
• Returns to baseline
Innate immune system
Adaptive immune system
Response in minutes to hours
Response in days to weeks
Recognizes broad classes of pathogens
Pathogen-specific response
No immunological memory
Exposure leads to immunological memory
Found in nearly all forms of life
Found only in jawed vertebrates
Key Players in Innate Immunity
Barriers (Skin, epithelia)
Enzymes,
Anti-microbial peptides
Microbial Recognition Receptors
Complement proteins
IMM/MICROM 441
Lecture #4 October 1/08
Kaja
Page 4 of 26
What influences adaptive immune
response to a vaccine?
- Innate immune responses shape the quantity, quality and longevity
of the adaptive response (Pulendran and Ahmed, Cell, 2006)
-Engineer a vaccine to induce desired innate responses
Vaccine vector
or adjuvant
Vaccine
antigen
http://research.dfci.harvard.edu/innate/images/innate/immunities.gif
Innate Immune Responses to Vaccination
• Few studies have examined innate responses in humans
after vaccination (Querec et al., NI, 2009; Gaucher et al., JEM, 2008)
• We implemented a clinical trial of an adenoviral vector
HIV vaccine (Merck Ad5):
1. Which systemic innate immune responses can be
measured in blood after vaccination by intramuscular
injection?
2. What are the optimal time points to identify these
innate responses in humans?
3. Which innate responses correlate with adaptive
responses?
Adenoviral vectors for
T-cell-based HIV vaccines
• Adenoviral vectors containing HIV inserts found to elicit
high-magnitude CD8+ T cell responses
~80% of vaccinees demonstrate T cell immunogenicity by ELISpot
~300 SFU/106 PBMC
-responses were durable
(Priddy et al., CID, 2008)
(Corey et al., AIDS 2009)
• Step Study
Innate Immune Responses:
HVTN 071 (n=35)
1.5 x 1010
genomes
MrkAd5
gag/pol/nef
Ad5 injection (IM):
Sampling points:
Day:
Samples:
0 6h 1
3
7
Whole blood (Flow cytometry)
Serum (Luminex)
PBMC RNA (Microarray)
n=11
28
PBMC (ICS)
Innate Immune Responses:
HVTN 071
1.5 x 1010
genomes
MrkAd5
gag/pol/nef
Ad5 injection (IM):
Sampling points:
Day:
Samples:
0 6h 1
3
n=24 (serum only)
7
28
PBMC (ICS)
PTID:
071-008
071-048
071-056
071-035
071-044
071-050
071-025
071-032
071-052
071-046
071-015
071-054
071-018
071-042
071-024
071-028
071-057
071-006
071-022
071-047
071-012
071-059
071-033
071-053
071-058
071-031
071-029
071-017
071-036
071-016
071-055
071-040
071-051
071-014
071-026
Ad5 serum neutralizing
antibody titer
Baseline Serum Ad5 Neutralizing Antibody Titers
10000
Baseline Ad5 Titer
1000
High, >1000
Moderate, 200-999
Low, 19-199
Negative, ≤18
100
10
Group A
Group B (pre-vaccine and day 1)
(n=11)
(n=24)
Rapid Changes in Peripheral Blood Cell Populations
after MRKAd5 gag/pol/nef Vaccination
Lymphocytes
4000
3000
1000
*
250
0
0
24
48
72
96 120 144 168
Granulocytes
6000
*
500
1000
0
*
0
24
48
72
96 120 144 168
Baseline Ad5 Titer
Moderate, 200-999
Low, 19-199
Negative, ≤18
4000
Cells/ml Blood
Monocytes
750
*
2000
1250
2000
p<0.05,
*
Timepoint vs.
0
0
24
48
72
96 120 144 168
Hours Post Vaccination
pre-vaccine (0 hrs.),
Wilcoxon signed rank test
Multiple Cell Types Exit the Blood
CD8 T cells
1000
*
500
CD4
cells
CD4+ TTcells
cells
CD19+B
Lymphocytes
2000
400
1500
300
1000
24
48
72
168
NK cells
800
600
Cells/ml Blood
100
0
0
400
*
200
0
0
24
48
72
Hours Post Vaccination
168
0
0
14
12
10
8
6
4
2
0
*
200
*
500
0
*
24
48
72
168
Plasmacytoid DC
0
24
48
72
168
0
24
48
72
168
Myeloid DC
35
30
25
20
15
10
5
0
0
24
48
72
168
Serum Proinflammatory Cytokines &
Chemokines Increase Rapidly after Vaccination
IFN-g
*
100
35
4
10
0
24
48
72
5
2
0
0
0
96 120 144 168
IP-10/CXCL-10
2000
1500
24
48
72
96 120 144 168
MCP-1/CCL2
2000
*
*
0
24
48
72
96 120 144 168
Baseline Ad5 Titer
Moderate, 200-999
Low, 19-199
Negative, ≤18
1500
1000
*
6
15
0
*
12
8
20
25
TNF-a
10
25
50
Serum pg/ml
*
30
75
500
IL-6
1000
*
*
*
0
p<0.05,
*
Timepoint vs.
500
0
0
24
48
72
96 120 144 168
Hours Post Vaccination
0
24
48
72
96 120 144 168
pre-vaccine (0 hrs.),
Wilcoxon signed rank test
Serum Anti-inflammatory Cytokines Also
Increase after MRKAd5 gag/pol/nef Vaccination
* IL-1Ra
5000
4000
IL-10
100
*
75
3000
*
Serum pg/ml
50
2000
25
1000
0
0
0
24
48
72
96 120 144 168
0
24
48
72
96 120 144 168
Hours Post Vaccination
Baseline Ad5 Titer
Moderate, 200-999
Low, 19-199
Negative, ≤18
p<0.05,
*
Timepoint vs.
pre-vaccine (0 hrs.),
Wilcoxon signed rank test
Summary of Significant Changes in Serum
Cytokines/Chemokines at 24 Hours
Cytokine/
Chemokine
IP-10/CXCL10
IL-1Ra
IL-10
IFN-γ
MCP-1/CCL2
IL-6
IL-7
TNFα
MIP-1β
Median Serum
Cytokine Levels (pg/ml)
n=35
Pre-vaccine 24 hours
70
350
5.4
0.1
180
4.3
7.1
5.1
120
630
1700
12
4.4
380
6.7
8.0
7.1
160
Median
Fold
Change
n=35
11
4.7
2.4
2.4
2.0
1.7
1.4
1.3
1.2
Other serum analytes that were measured,
but did not change at 24 hours:
EGF, Eotaxin, Fractalkine, G-CSF, GM-CSF, IFN-a, IL-1a, IL-1b, IL-2,
IL-4, IL-5, IL-8, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, MIP-1a, sCD40L,
TGF-a, VEGF
Ad5 Negative/Low Titer Individuals
Show Significantly Greater Changes in
Serum Cytokines/Chemokines at 24 Hours
Cytokine/
Chemokine
MCP-1
IP-10
IL-1Ra
Median Serum
Cytokine Levels (pg/ml)
At 24 Hours Post
Vaccination
Ad5
negative/low
n=27
Ad5
medium/high
n=6
p-value
(Wilcoxon
Rank Sum
Test on log
fold change
relative to
prevaccine)
420
720
2100
260
180
870
0.002
0.02
0.02
PBMC Gene Expression
PBMC
Isolate RNA
Affymetrix
human exon arrays
(10 people; 5 timepoints
Analysis
(collaboration
with Dan Zak, ISB)
• Discover other innate
immune responses to Ad5
vaccination
• Identify additional differences
in responses between Ad5
naïve and Ad5 immune
individuals
PBMC Microarrays Show Many Genes with
Significant* Temporal Expression Responses
Med
Ad5 Titer
Low
Med
Ad5 Titer Ad5 Titer
volunteer ID:
timepoint:
*p<10-6, 1 way ANOVA
Up-regulated
Down-regulated
What do the gene
expression changes mean?
• 2 sources of changes in gene expression:
– Changes in the PBMC composition
– Changes in the response of cells that are in
the blood to the vaccine
• Attenuated gene expression changes in
Ad5 medium titer subjects
– are these individuals effectively exposed to a
lower vaccine dose?
6hrs: 1 gene down-regulated
(p<0.001, |log2(FC)| > 0.5)
Innate DB: www.innatedb.ca/
6hrs: 1 gene down-regulated
(p<0.001, |log2(FC)| > 0.5)
CCL3
MIP-1a
24hrs: 1065 genes up/down-regulated
(p<0.001, |log2(FC)| > 0.5, FDR = 0.01)
24hrs: TLR pathway up-regulated
(p<0.001, |log2(FC)| > 0.5, FDR = 0.01)
24hrs: STAT1 targets/interactors upregulated (p<0.001, |log2(FC)| > 0.5, FDR = 0.01)
24hrs: TCR pathway down-regulated
(p<0.001, |log2(FC)| > 0.5, FDR = 0.01)
72hrs: 116 genes up/down-regulated
(p<0.001, |log2(FC)| > 0.5, FDR = 0.02)
72hrs: 116 genes up/down-regulated
(p<0.001, |log2(FC)| > 0.5, FDR = 0.02)
CSF1R
CSF1R
168hrs: 11 genes up/down-regulated
(p<0.001, |log2(FC)| > 0.5, FDR = 0.2)
ENDO-PDI
TXNDC5
TXNDC5 = EndoPDI: a protein preferentially expressed in endothelial
cells that acts as a stress survival factor during hypoxia
(Sullivan et al., 2003)
Summary
• MRK Ad5 gag/pol/nef administered IM induces robust
innate immune responses in the blood:
– rapid influx of monocytes/mDC into the blood and efflux of
lymphocytes/pDC from the blood
• trafficking to and from the site of vaccination?
– changes in serum chemokines and PBMC gene expression indicate
PBMC are the source of many serum factors
– maximal responses occur at 24hrs post-vaccination
• Pre-existing neutralizing antibodies to Ad5 attenuate the
innate response
– reduced production of MCP-1, IP-10 and IL-1Ra
– muted gene expression changes in PBMC
• Potential correlates for magnitude of CD8 response are
being identified
Future Directions
• Compare innate responses to Mrk rAd5 vaccine with
responses to other vaccines
– Other candidate HIV vaccines (e.g. MVA: HVTN 908)
– Vaccines currently in use (e.g. YFV)
• Evaluate innate immune responses to vaccines at
mucosal sites to determine effects at the site of HIV
acquisition
• Develop an in vitro system to test effects of identified
innate response genes on the adaptive response
Acknowledgements
Julie McElrath
Steve De Rosa
Eric Peterson
Joanne Chang
Sam Pine
Greg Spies
Don Carter
Olivier Defawe
Mingchao Shen
Reneé Ireton
Phyllis Stegall
HVTN Laboratory Program
HVTU
(Seattle, Rochester, Nashville, Birmingham)
Ann Duerr
Larry Corey
Jim Kublin
Volunteers
Merck
Michael Robertson
NIAID/DAIDS
Margaret Johnston
Dan Zak
Alan Aderem
UW STD/AIDS Research
Training Fellowship
CAVD Investigators
Joleen Borgerding
Nidhi Kochar
Liza Noonan
Yunda Huang
Li Qin
Steve Self
Innate immune detection of adenovirus
(Gilliet et al., 2008)
The Step Study
• 3000-person HIV vaccine trial started in late
2004
• Phase IIB test-of-concept study to establish
whether MRKAd5 expressing HIV-1 gag/pol/nef
could lower:
– HIV-1 infection rates
– Plasma viremia
• Interim analysis performed when 30 per-protocol
HIV infections occurred
HIV Incidence
(% per year)
Step Study Results
Overall
• No effect of the vaccine
on viral load
HIV Incidence
(% per year)
0-26
26-52
52-78
• Increased HIV infection in
vaccinees that had preexisting immunity to Ad5
Ad5≤18
HIV Incidence
(% per year)
0-26
26-52
52-78
• All further vaccinations
were halted
Ad5>18
0-26
26-52
52-78
Time Interval (weeks)
(Buchbinder et al., 2008)
• Mechanisms under
investigation
(Buchbinder et al., 2008; McElrath et al., 2008)