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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+