2014 “Towards an HIV Cure” symposiumMelbourne

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

EuroNeut-41EuroNeut-41

INSERM U1109, Université de Strasbourg, Strasbourg, FRANCE

The role of dendritic cells (DCs) in HIV infection and dissemination

Function of DCs : Antigen Presentation

Mucosal surface (genital mucosa)

HIV infection

Transmission

HIV

Lesion

Langerhans cellSubmucosal epithelium

Mucous layer

Macrophages

CD4 T-cells

HIV cell-free infection

Myeloid Dendritic Cells

HIV cell-to-cell transmission

PlasmacytoidDCs (pDCs)

pDC-mediated transfer

DisseminationLymphoid tissues

«  Virological Synapse »

DCsT-cells

(Piguet and Sattentau, J Clin Invest. 2004)

(Su B and Moog C, Front. Immunol 2014)

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)

pDCs efficiently transfer HIV-1 to CD4 T-cells (Evans et al., Retrovirology 2011; Lore et al., J Exp Med 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)

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

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)

Intracellular SAMHD1 expression : anti-SAMHD1 Ab

ELISA test for type 1 IFN production

HIV-1-loaded pDCs + CD4 T-cells -> Mimic early mucosal transmission of HIV-1 infection

Flow Cytometry

Primary R5 or Transmitted/Founder (T/F) HIV-1-loaded primary pDCs

(2h of infection)

bNAbs(VRC01, PGT121)

+/-

72h post-infection

SIV-Vpx-VSV-G(VLP-Vpx)

+/-

Autologous PHA/IL-2 activated CD4 T lymphocytes

Extensive wash

+/-

Indinavir (IDV)(protease inhibitor)

+/-

ELISA

SAMHD1+

Gag

+

Stimulation of HIV-1 replication in cocultured pDCs with CD4 T lymphocytes

pDCs:

CD4 T-cells:

HIV-1BaL:

AZT (5 µM):

+ +

- -+ +

+

CD

123+

pDC

s

Intracellular viral Gag antigen (p24)

-3% 0.2%

+ + + +- ++ -

8%

3.6% 0.1%

< 0.1%

< 0.1%

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

0

4

8

12

16

0

300

600

900

1200

Coculture with CD4 T lymphocytes enhances HIV-1 replication in primary pDCs

% of Infected CD123+ pDCs

+VLP-Vpx:

HIV-1BaL: +

+--CD4 T-cells: +

+- -

(72h PI, HIV-1BaL, n = 3 - 9 donors) (means±SEM, Two-tailed Paired t test)

Downregulation of SAMHD1 in pDC

cocultured with CD4 T lymphocytes

was associated with an increased

HIV-1 in cocultured pDCs.

***

N.S.

Median Fluorescence Intensity (MFI) in

CD123+ pDCsSAMHD1

*N.S.

Vpx of HIV-2 and SIVmac can degrade

SAMHD1 in mDCs (Laguette et al., Nature

2011)

Two modes of HIV-1 transfer describedin trans and in cis

Trans-infection

CCR5CD4

Dendritic cells

Receptor

Cis-infection

bNAbs inhibit HIV-1 replication of free virus particles

MVBVirological Synapse

Exosome

Lyse

Production of new virion

Infected DCs

bNAbs

X X XX

XX

X

X

X

X

Inhibitory activity of bNAbs on T/F HIV-1 transmission to T-cells ?

CD4 T-cells

Protease inhibitors prevent maturation of new virions and cis-infection

Single cycle of T/F HIV-1Bx11 infection ?

In pDCs: single cycle of infection

72h post-infection

In CD4 T-cells: 51% of infection

corresponds to HIV-1 transfer in

trans from pDCs and 49% of

infection corresponds to cis-

infection.0

102030405060708090

100

Infe

cted

Cel

ls (%

of c

ontr

ol)

IDV (1 µM): - +

Infected pDC: CD4 T-cells:

+ + + ++ + + +- +

pDCs T-cells

In the coculture (Infected pDC + CD4 T-cells)

Protease inhibitor Indinavir (IDV):Prevent final assembly and maturation of new virions

(72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)

N.S. *

51% trans-infection

49% cis-infection

T/F HIV-1Bx11

To investigate the inhibition of T/F HIV-1 transfer

in trans from pDCs to CD4 T-cells by bNAbs

0102030405060708090

100

HIV-1 bNAb VRC01 inhibits HIV-1 transfer from pDCs to CD4 T lymphocytes

Infe

cted

Cel

ls (%

of c

ontr

ol)

-VRC01 (µg/ml):

In the coculture (Infected pDC + T-cells)

Cell-to-Cell Transmission

20 2

Infected pDC T-cells Infected T-cells

Neutralization Cell-Free Infection

- 20 2 - 20 2

(72h PI, HIV-1Bx11, n = 4 donors) (means±SEM, Two-tailed Paired t test)

N.S. *N.S. **

***

N.S.

N.S.T/F HIV-1Bx11

bNAb: VRC01

0102030405060708090

100

+20-

+ +20-

+ +20-

+In

fect

ed C

ells

(% o

f con

trol

)

VRC01 (µg/ml):IDV (1 µM):

In the coculture (Infected pDC + T-cells)Cell-to-Cell Transmission

Infected pDC T-cells Infected T-cells

NeutralizationCell-Free Infection

Trans-infection

N.S. * *N.S.

T/F HIV-1 replication in pDCs slightly decreased

when VRC01 was added to pDCs 2h after infection

T/F HIV-1 transfer to CD4 T-cells was prevented

by 80% (20 µg/ml VRC01) and 35% (2 µg/ml VRC01)

VRC01 inhibited HIV-1 transfer with a similar

efficiency as cell-free infection of CD4 T-cells

We found a similar inhibition of T-cell infection by VRC01, demonstrating its capacity to inhibit HIV-1 trans-infection as well as further transfer in cis to T-cells

HIV-1 bNAb PGT121 inhibits HIV-1 transfer from pDCs to CD4 T lymphocytes

Infe

cted

Cel

ls (%

of c

ontr

ol)

-PGT121 (µg/ml):

In the coculture (Infected pDC + T-cells)

Cell-to-Cell Transmission

5

Infected pDC T-cells Infected T-cells

Neutralization Cell-Free Infection

(72h PI, HIV-1Bx11, one representative experiment was shown)

0102030405060708090

100

+5-

+ +5-

+ +5-

+In

fect

ed C

ells

(% o

f con

trol

)

PGT121 (µg/ml):IDV (1 µM):

In the coculture (Infected pDC + T-cells)Cell-to-Cell Transmission

Infected pDC T-cells Infected T-cells

NeutralizationCell-Free Infection

Trans-infection

0102030405060708090

100

- 5 - 5

PGT121 also inhibited HIV-1 transfer and cell-free infection of CD4 T lymphocytes

T/F HIV-1Bx11

bNAb: PGT121

Induction of pDC maturation in the presence of CD4 T lymphocytes

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

VRC01 (µg/ml):

pDCs + CD4 T-cells

20

IDV (1 µM):

HIV-1Bx11:202

pDCs

+ + +---

--

+---

--

+ +-

- - + +

05

1015202530

% C

D83

+ C

D12

3+ p

DC

s*

N.S. (1-way ANOVA, Kruskal-Wallis test)

**

T/F HIV-1Bx11

(72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)

050

100150200250300350

Increase of IFN-α production by infected pDCs in the presence of CD4 T lymphocytes

IFN

-α (%

of c

ontr

ol)

VRC01 (µg/ml):

pDCs + CD4 T-cells

20

IDV (1 µM):

HIV-1Bx11:202

pDCs

+ + +---

--

+---

--

+ +-

- - + +

IFN-α was induced following HIV-1 infection and was significantly increased by CD4 T-cells

IFN-α induction was not inhibited following VRC01 inhibition of HIV-1 or IDV treatment

T/F HIV-1Bx11 N.S. (1-way ANOVA, Kruskal-Wallis test)

*

(72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)

IFN-α between

10 ~ 200 ng/ml

The increased immune sensing and pDC maturation during pDC/lymphocyte cross talk

might promote efficient innate immune responses and may be able to control viral

infection

Mucosal surface (genital mucosa)

HIVLesion

Langerhans cell

Submucosal epithelium

Mucous layer

pDCs

Conclusion and Highlights

pDC-mediated transfer

HIV infection

Transmission

DisseminationHIV Cell-Free Infection

Exosome

Virologicalsynapse

HIV Cell-to-Cell Transmission

pDCs poorly support viral replication. However, in a physiologically relevant coculture model, HIV-1 replication increases in pDCs cocultured with T-cells pDCs efficiently transfer HIV-1 to adjacent CD4 T lymphocytes

bNAbs should be induced by vaccination directly at mucosal site to prevent the early dissemination of HIV-1 after sexual transmission

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

Acknowledgements

INSERM U1109, FMTS, Strasbourg, France"Neutralization" Team

Alexandre Lederle Géraldine Laumond Camille Ducloy Sylvie Schmidt Thomas Decoville Christiane Moog

Olivier Schwartz (anti-SAMHD1 Ab & VLP-Vpx)

EuroNeut-41EuroNeut-41

Pasteur Institute, Paris, FranceVirus and Immunity Unit

John Mascola (VRC01, NIH, Bethesda, MD)

Dennis Burton & Pascal Poignard (PGT121, Scripps Research Institute, La Jolla, CA)

Antibodies obtained from :

Gating strategy for detection HIV-1 replication

FSC

SSC

CD3+

SSC

Live/Dead

SSC

CD123+ Live/Dead

Living CD123+ pDC

Living CD3+ CD4 T cells

SSC

SSC p24+C

D12

3+

SAMHD1+

CD

123+

SAMHD1+

p24+