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2014 “Towards an HIV Cure” symposium
Melbourne
Potent and Broadly Anti-HIV-1 Neutralizing Antibodies Inhibit
HIV-1 Transmission from Plasmacytoid Dendritic Cells to
CD4 T Lymphocytes
Bin Su, Alexandre Lederle, Géraldine Laumond, Camille Ducloy,
Sylvie Schmidt, Thomas Decoville, and Christiane Moog
INSERM U1109, Université de Strasbourg, Strasbourg, FRANCE
The role of dendritic cells (DCs) in HIV
infection and dissemination
HIV
Function of DCs :
Antigen Presentation
Lesion
Mucous layer
Mucosal surface
(genital mucosa)
Submucosal epithelium
Langerhans cell
Myeloid Dendritic Cells
HIV infection
CD4 T-cells
Transmission
Macrophages
Plasmacytoid
DCs (pDCs)
pDC-mediated transfer
HIV cell-free infection
HIV cell-to-cell transmission
« Virological
Synapse
(Su B and
Moog C, Front.
Immunol»2014)
Dissemination
Lymphoid tissues
DCs
T-cells
(Piguet and Sattentau, J Clin Invest. 2004)
Plasmacytoid dendritic cells (pDCs) in
HIV infection and antibodies
 pDCs link innate/adaptive immunity (Fitzgerald-Bocarsly et al., JLB 2010; Liu, Annu Rev Immunol 2005)
 HIV-1 replicates poorly in pDCs due to expression of the host restriction factor SAMHD1
(Bloch et al., AIDS Res Hum Retroviruses 2014; Laguette et al., Nature 2011)
 pDCs efficiently transfer HIV-1 to CD4 T-cells (Evans et al., Retrovirology 2011; Lore et al., J Exp Med 2005)
 Novel broadly neutralizing antibodies (bNAbs) such as VRC01 potently inhibit most
strains of HIV-1 (> 91% viruses), mimics binding of CD4 to gp120 (Wu et al., Science 2010)
 VRC01 prevents infection in SHIV macaque models and it may be able to prevent
infection in humans (Pegu et al., Sci Transl Med 2014; Shingai et al., Nature 2013; Pegu et al., J Immunol 2011)
Objectives :
Analyze the ability of bNAbs to inhibit HIV-1 transmission from primary pDCs to
autologous CD4 T lymphocytes
Methods : HIV-1 transfer assay
HIV-1-loaded pDCs + CD4 T-cells -> Mimic early mucosal transmission of HIV-1 infection
SIV-Vpx-VSV-G
(VLP-Vpx)
+/-
Extensive wash
Indinavir (IDV)
bNAbs
(VRC01, PGT121) (protease inhibitor)
+/-
+/-
Flow
Cytometry
72h post-infection
ELISA
Primary R5 or Transmitted/Founder (T/F)
HIV-1-loaded primary pDCs
(2h of infection)
+/-
Autologous PHA/IL-2 activated
CD4 T lymphocytes
Characterization of the cells : CD123+ pDCs, CD3+ CD4 T-cells; CD83+ / CD86+ pDC maturation
HIV-1 replication or inhibition by bNAbs : intracellular viral Gag antigens (p24)
ELISA test for type 1 IFN production
Gag+
Intracellular SAMHD1 expression : anti-SAMHD1 Ab
SAMHD1+
Stimulation of HIV-1 replication in
cocultured pDCs with CD4 T lymphocytes
pDCs:
+
CD4 T-cells:
-
HIV-1BaL:
+
AZT (5 µM):
+
+
+
-
-
CD123+ pDCs
3%
+
+
+
+
+
+
-
8%
0.2%
< 0.1%
3.6%
0.1%
< 0.1%
Intracellular viral Gag antigen (p24)
 Increased HIV-1 replication in pDCs in the presence of autologous CD4 T lymphocytes
 HIV-1 restriction in pDCs may be overcome under coculture conditions
Coculture with CD4 T lymphocytes enhances
HIV-1 replication in primary pDCs
N.S.
16
***
12
% of Infected
CD123+ pDCs
 Downregulation of SAMHD1 in pDC
8
cocultured with CD4 T lymphocytes
4
was associated with an increased HIV-1
in cocultured pDCs.
0
N.S.
*
1200
Median Fluorescence
Intensity (MFI) in
CD123+ pDCs
SAMHD1
900
600
 Vpx of HIV-2 and SIVmac can degrade
300
SAMHD1 in mDCs (Laguette et al., Nature 2011)
0
HIV-1BaL:
CD4 T-cells:
VLP-Vpx:
+
-
+
+
-
+
-
+
(72h PI, HIV-1BaL, n = 3 - 9 donors) (means±SEM, Two-tailed Paired t test)
Two modes of HIV-1 transfer described
in trans and in cis
bNAbs
CCR5
CD4
Receptor
X
X
XX
Exosome
X
MVB
Virological
Synapse
X
X
X X
Dendritic cells
Lyse
CD4 T-cells
Production of
new virion
X
Trans-infection
Infected DCs
Cis-infection
 Protease inhibitors prevent maturation of new virions and cis-infection
 bNAbs inhibit HIV-1 replication of free virus particles
 Inhibitory activity of bNAbs on T/F HIV-1 transmission to T-cells ?
Single cycle of T/F HIV-1Bx11 infection ?
T/F HIV-1Bx11
pDCs
T-cells
N.S.
*
Infected Cells (% of control)
100
Protease inhibitor Indinavir (IDV):
Prevent final assembly and
maturation of new virions
90
80
49% cis-infection
70
 In pDCs: single cycle of infection
72h post-infection
60
50
40
 In CD4 T-cells: 51% of infection
30
51% trans-infection
20
from pDCs and 49% of infection
10
0
Infected pDC:
CD4 T-cells:
IDV (1 µM):
corresponds to HIV-1 transfer in trans
corresponds to cis-infection.
+ +
+ +
- +
+ +
+ +
- +
 To investigate the inhibition of T/F HIV-1 transfer
in trans from pDCs to CD4 T-cells by bNAbs
In the coculture
(Infected pDC + CD4 T-cells)
(72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)
HIV-1 bNAb VRC01 inhibits HIV-1 transfer
from pDCs to CD4 T lymphocytes
T/F HIV-1Bx11
Trans-infection
N.S.
bNAb: VRC01
N.S.
N.S.
N.S.
*
N.S.
*
**
100
**
Infected Cells (% of control)
Infected Cells (% of control)
N.S.
90
80
when VRC01 was
70 added to pDCs 2h after infection
80
70
60
50
 T/F HIV-1 transfer
to CD4 T-cells was prevented
60
50
40
by 80% (20 µg/ml VRC01) and 35% (2 µg/ml VRC01)
40
30
20
30
20
10
 VRC01 inhibited
HIV-1 transfer with a similar
10
VRC01
(µg/ml):
*
 T/F HIV-1 replication in pDCs slightly decreased
90
0
*
100
0
efficiency as cell-free infection of CD4 T-cells
-
20 2
Infected pDC
-
20 2
T-cells
In the coculture
(Infected pDC + T-cells)
-
VRC01 (µg/ml):
20 2
Infected T-cells
IDV (1 µM):
-
20
+ +
Infected pDC
-
20
+ +
T-cells
-
20
+ +
Infected T-cells
In the coculture
(Infected
pDC + T-cells) its capacity
Neutralization
 We found a similar inhibition of T-cell infection by VRC01,
demonstrating
to
Cell-Free
Infection
Cell-to-Cell
Transmission as well as further transfer
inhibit
HIV-1 trans-infection
in cis toTransmission
T-cells
Cell-to-Cell
Cell-Free Infection
Neutralization
(72h PI, HIV-1Bx11, n = 4 donors) (means±SEM, Two-tailed Paired t test)
HIV-1 bNAb PGT121 inhibits HIV-1 transfer
from pDCs to CD4 T lymphocytes
T/F HIV-1Bx11
Trans-infection
100
Infected Cells (% of control)
bNAb: PGT121
90
Infected Cells (% of control)
 PGT121
also inhibited HIV-1 transfer and cell-free infection of CD4 T lymphocytes
100
90
80
70
60
50
40
30
20
10
0
PGT121
(µg/ml):
80
70
60
50
40
30
20
10
0
-
5
Infected pDC
-
5
T-cells
In the coculture
(Infected pDC + T-cells)
Cell-to-Cell Transmission
-
PGT121 (µg/ml):
-
5
-
5
-
5
IDV (1 µM):
+
+
+
+
+
+
5
Infected T-cells
Infected pDC T-cells
Infected T-cells
Neutralization
In the coculture
(Infected pDC + T-cells)
Neutralization
Cell-Free Infection
Cell-to-Cell Transmission Cell-Free Infection
(72h PI, HIV-1Bx11, one representative experiment was shown)
Induction of pDC maturation in the presence of
CD4 T lymphocytes
T/F HIV-1Bx11
% CD83+ CD123+ pDCs
N.S. (1-way ANOVA, Kruskal-Wallis test)
30
**
25
20
*
15
10
5
0
HIV-1Bx11:
VRC01 (µg/ml):
IDV (1 µM):
- +
- - pDCs
- +
- - -
+ + + +
20
2
-
20
- - + +
pDCs + CD4 T-cells
 Infection with HIV-1Bx11 did not induce pDC maturation
 Maturation markers were increased in the context of coculture with CD4 T-cells ± HIV-1
and independently of VRC01 or IDV treatment
(72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)
Increase of IFN-α production by infected pDCs
in the presence of CD4 T lymphocytes
T/F HIV-1Bx11
IFN-α between
10 ~ 200 ng/ml
IFN-α (% of control)
N.S. (1-way ANOVA, Kruskal-Wallis test)
*
350
300
250
200
150
100
50
0
HIV-1Bx11:
VRC01 (µg/ml):
IDV (1 µM):
- +
- - pDCs
- +
- - -
+ + + +
20
2
-
20
- - + +
pDCs + CD4 T-cells
IFN-α
was induced
following
HIV-1and
infection
and was significantly
increased by CD4
T-cells
The increased
immune
sensing
pDC maturation
during pDC/lymphocyte
cross
talk
efficient
innate
immune
responses
and
may be of
able
to control
viral infection
might
IFN-αpromote
induction
was not
inhibited
following
VRC01
inhibition
HIV-1
or IDV treatment
(72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)
Conclusion and Highlights
Mucosal surface
(genital mucosa)
HIV
Lesion
Mucous layer
 pDCs poorly support viral replication. However, in a physiologically relevant
cocultureLangerhans
model,cell
HIV-1 replication increases in pDCs cocultured with T-cells
 pDCs efficiently transfer HIV-1 to adjacent CD4 T lymphocytes
Submucosal epithelium
pDC-mediated
transfer
pDCs
 bNAbs VRC01 & PGT121 prevent HIV-1 transmission with a similar efficiency as
cell-free infection of T-cells, and do not impair innate immune sensing of HIV-1
 bNAbs should be induced by vaccinationVirological
directly at mucosal site to prevent the
synapse
HIVearly
infection
dissemination of HIV-1 after sexual transmission
Transmission
Exosome
Dissemination
HIV Cell-to-Cell Transmission
HIV Cell-Free Infection
Acknowledgements
INSERM U1109, FMTS, Strasbourg, France
"Neutralization" Team
Alexandre Lederle
Géraldine Laumond
Camille Ducloy
Sylvie Schmidt
Thomas Decoville
Christiane Moog
Pasteur Institute, Paris, France
Virus and Immunity Unit
Olivier Schwartz (anti-SAMHD1 Ab & VLP-Vpx)
Antibodies obtained from :
John Mascola (VRC01, NIH, Bethesda, MD)
Dennis Burton & Pascal Poignard (PGT121,
Scripps Research Institute, La Jolla, CA)
Gating strategy for detection HIV-1 replication
CD123+
Living CD123+ pDC
CD123+
Live/Dead
SSC
Living CD3+ CD4 T cells
FSC
CD123+
SSC
SSC
p24+
CD3+
Live/Dead
p24+
SSC
SSC
SAMHD1+
SAMHD1+