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Anti-HIV Antibody Responses
Reflect the Quantifiable
HIV Reservoir Size
Sulggi Lee, M.D., Ph.D.
Assistant Professor of Medicine
University of California, San Francisco
July 20, 2015
Chronic/Latent Viral Infections Lead to
Prolonged Antibody Responses
VZV Antibody Half-Life ~ 50 Years
Amanna NEJM 2007
(Infectious Units per 106 cells)
Latently Infected Cells
The Size of the HIV Reservoir is
Also Stable During Long-Term ART
10000
Time to eradication
> 73.4 years
1000
100
10
1
0.1
-
0.01
0.001
0.0001
0.00001
0
1
2
3
4
5
6
7
Time on HAART (years)
Siliciano Nat Med 2003
Anti-HIV Antibody Levels May Distinguish
Individuals With Different HIV Reservoir Sizes
Anti-gp41 Levels
*
*
*
Light Units (LU)
Antibody Levels
*
Burbelo JID 2014
Anti-HIV Antibody Levels May Be Sensitive
Measures of the Reservoir Size Over Time
Median gp41 Levels by Years of ART
Anti –gp41 Levels
(LU)
Light Units
Light Units
Antibody Levels
1.0×1007
Median
1.0×1006
0
00
1
2
3
4
5
6
Duration of ART (years)
Years
of ART
Pre-ART
Longitudinal data from Burbelo JID 2014
Hypothesis: Antibody Levels in Blood at
Steady-State Reflect Total Body Reservoir Size
Primary Infection
Antibody Production
CD4+
Y
Establishment of
Infection in
Lymphoid Tissue
Current Issues in Measuring HIV Reservoir
• How to measure the success of HIV cure treatments if
no scalable, precise assay of the reservoir?
• The HIV reservoir is much larger in tissue than in blood
– A reliable measure that reflects the HIV reservoir in tissue?
• Most measures of the HIV reservoir are virologic assays,
not cell-based assays measuring host response to virus
Eriksson PLoS Pathogens 2013, Ho Cell 2013, Chun JID 2008, Yukl JID 2010
Study Design
• Aim: Determine whether anti-HIV antibody
levels reflect the size of the latent reservoir
• Participants: 61 HIV+ individuals who
– Initiated ART during chronic infection
– Have been ART-suppressed for at least 3 years
Novel Luciferase Immunoprecipitation Systems
(LIPS) Assay Detects Conformational Antibodies
One letter code for amino acids
Luciferase
P I V Q N I Q G Q M V H Q A I S P R T L N A W V K V V E E K A F S P E V I
P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M L K E
T I N E E A A E W D R V H P V H A G P I A P G Q M R E P R G S D I A G T T
S T L Q E Q I G W M T N N P P I P V G E I Y KR W I I L G L N K I V R M Y S
P T S I L D I R Q G P K E P F R D Y V D R F Y K T L R A E Q A S Q E V K N W
M T E T L L VQ N A N P D C K T I L K A L G P A A T LE E M M T A C Q G V
G G P G H K A R V L*
L
C
L
Luciferase
C
L = linear epitope
C= conformational epitopes
L
C
Question: Are HIV Reservoir Measures
Associated with Anti-HIV Antibody Levels?
12 Measures of the HIV Reservoir
• Total HIV DNA
•
•
•
CD4+ T cells (rtPCR)
PBMCs (ddPCR)
Resting CD4+ T cells (ddPCR)
• Integrated HIV DNA
•
•
•
CD4+ T cells (rtPCR)
PBMCs (aluPCR)
Resting CD4+ T cells (aluPCR)
• 2-LTR HIV DNA
•
•
•
CD4+ T cells (rtPCR)
PBMCs (ddPCR)
Resting CD4+ T cells (ddPCR)
7 Anti-HIV Antibody
Levels
•
•
•
•
•
•
•
gp120
gp41
reverse transcriptase
integrase
protease
matrix
p24
• Unspliced HIV RNA
•
CD4+ T cells (rtPCR)
Permutation Testing
• Plasma HIV RNA
•
Plasma (rtPCR single copy assay)
• Infectious Units per Million
•
Resting CD4+ T cells (VOA)
To test the association between
reservoir and antibody measures,
adjusted for multiple comparisons
Erikkson PLoS Pathogens 2013, Lewin J Virol 1999, Burbelo JID 2014
Study Participant Characteristics
Characteristics
N=61
Gender (Male)
59 (96%)*
Age (years)
56 (50-61)
Pre-ART HIV RNA (log10copies/mL)
3.9 (3.5-4.1)
Nadir CD4+ T cell count (cells/mm3)
215 (124-343)
Duration of ART (years)
6 (3-11)
Proximal CD4+ T cell count (cells/mm3)
670 (541-809)
*Number (%)
Median and Interquartile Range shown
Measures of the HIV DNA (Total, Integrated)
are Correlated with Anti-HIV Antibody Levels
R > 0.7
R > 0.5
R > 0.4
Env
Gag
Pol
gp120 gp41
RT
INT
PR
MA
p24
Total HIV DNA
CD4+ T cells (rtPCR)
0.16
-0.03
0.16
0.18
0.17
0.11
-0.08
PBMCs (ddPCR)
0.44
0.48
0.30
0.40
0.40
0.23
0.16
Resting CD4+ T cells (ddPCR)
0.51
0.28
0.62
0.57
0.60
0.54
0.29
CD4+ T cells (rtPCR)
0.21
0.07
0.20
0.11
0.27
0.06
0.06
PBMCs (aluPCR)
0.80
0.73
0.76
0.70
0.54
0.53
0.41
Resting CD4+ T cells (aluPCR)
0.50
0.72
0.82
0.50
0.25
0.34
0.25
Integrated HIV DNA
Measures of the “Active” or Replication-Competent
Reservoir are Weakly Correlated with Anti-gp120 Levels
R > 0.7
R > 0.5
R > 0.4
Env
Gag
Pol
gp120 gp41
RT
INT
PR
MA
p24
2-LTR HIV DNA
CD4+ T cells (rtPCR)
0.05
-0.12
-0.10
-0.10
0.14
0.17
-0.07
PBMCs (ddPCR)
0.47
0.26
0.33
0.56
0.46
0.39
0.01
Resting CD4+ T cells (ddPCR)
-0.05
0.14
0.09
0.01
0.02
0.09
-0.14
0.40
0.15
0.09
0.12
0.05
0.07
0.10
-0.03
-0.24
-0.15
-0.10
-0.16
-0.06
-0.01
0.45
0.43
0.20
0.31
0.30
0.25
0.02
Unspliced HIV RNA
CD4+ T cells (rtPCR)
Plasma HIV RNA
Plasma (rtPCR SCA)
Infectious Units (Per 106)
Resting CD4+ T cells (VOA)
Overall, HIV Reservoir Measures Strongly
Correlate with Anti-gp120, -gp41, and -RT Levels
Env
Gag
Pol
gp120
gp41
RT
INT
PR
MA
p24
0.80
0.73
0.82
0.70
0.60
0.54
0.41
0.009
0.04
0.007
0.05
0.20
0.34
0.68
All HIV Reservoir Measures
Correlation (R)
P
* Adjustment for age, nadir CD4+ T cell count, proximal CD4+ T cell count, years of
ART suppression, pre-ART viral load did not significantly alter these results.
Conclusions
• We observed a strong association between measures
of the latent HIV reservoir and anti-HIV antibody levels
• Overall, anti-gp120, -gp41, and -RT were most strongly
correlated with HIV reservoir size
• Total and integrated HIV DNA were strongly associated
with antibody levels against Env and Pol proteins
• Measures of the “active” (CA-US RNA, 2-LTR DNA) and
replication-competent (VOA) reservoir were weakly
associated with anti-gp120 levels
Remaining Questions
• Can viral RNA or proteins be produced in cells
with defective proviruses?
– And elicit a host immune response?
• Is there preferential B cell response?
– Relative “importance” of Env > Pol > Gag antigens?
• Why are HIV CA-DNA and CA-RNA but not plasma
RNA correlated with anti-HIV antibody levels?
– Do antibody levels do a good job of quantifying the
HIV tissue reservoir?
Can Anti-HIV Antibody Levels Monitor
Response to HIV Cure Strategies?
“kick”
HIV DNA
HIV proteins
HIV US RNA
HIV virions
Latent infec on
Antibody Levels Pre-Treatment
Cell death
Antibody Levels Post-Treatment
Acknowledgements
• UCSF
– Steven Deeks
– Saunak Sen
• NIH/NIDCR
– Peter Burbelo
• Johns Hopkins
– Robert Siliciano
– Janet Siliciano
• University of Melbourne
– Sharon Lewin
• UCSD
– Douglas Richman
– Matthew Strain
• University of Pennsylvania
– Una O’Doherty
• University of Sydney
– Sarah Palmer
• University of Montreal
– Nicolas Chomont
– Remi Fromentin