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2014 “ Towards an HIV Cure ” symposium Melbourne

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.

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2014 “ Towards an HIV Cure ” symposium Melbourne

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  1. 2014 “Towards an HIV Cure” symposiumMelbourne Potent and Broadly Anti-HIV-1 Neutralizing Antibodies Inhibit HIV-1 Transmission from PlasmacytoidDendritic Cells to CD4 T Lymphocytes Bin Su, AlexandreLederle, GéraldineLaumond, Camille Ducloy, Sylvie Schmidt, Thomas Decoville, and Christiane Moog INSERM U1109, Université de Strasbourg, Strasbourg, FRANCE

  2. Function of DCs : AntigenPresentation The role of dendriticcells (DCs) in HIV infection and dissemination HIV Lesion Mucous layer Mucosal surface (genitalmucosa) Submucosalepithelium Langerhanscell MyeloidDendriticCells HIV infection Transmission CD4 T-cells Macrophages Plasmacytoid DCs (pDCs) pDC-mediatedtransfer HIV cell-free infection HIV cell-to-cell transmission «  Virological Synapse » (Su B and Moog C, Front. Immunol 2014) Dissemination DCs Lymphoid tissues T-cells (Piguet and Sattentau, J Clin Invest. 2004)

  3. Plasmacytoiddendriticcells (pDCs) in HIV infection and antibodies • pDCs link innate/adaptive immunity (Fitzgerald-Bocarslyet 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; Laguetteet 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(Peguet al., SciTransl Med 2014; Shingaiet al., Nature 2013; Peguet al., J Immunol 2011) Objectives : Analyze the ability of bNAbs to inhibit HIV-1 transmission from primary pDCs to autologous CD4 T lymphocytes

  4. Methods : HIV-1 transfer assay HIV-1-loaded pDCs + CD4 T-cells -> Mimicearlymucosal transmission of HIV-1 infection Indinavir (IDV) (protease inhibitor) bNAbs (VRC01, PGT121) SIV-Vpx-VSV-G (VLP-Vpx) +/- +/- +/- Flow Cytometry Extensive wash 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) Intracellular SAMHD1 expression : anti-SAMHD1 Ab Gag+ ELISA test for type 1 IFN production SAMHD1+

  5. Stimulation of HIV-1 replication in coculturedpDCswith CD4 T lymphocytes + + + pDCs: CD4 T-cells: HIV-1BaL: AZT (5 µM): + - + + + - + + + - - - + 0.2% 3% 8% < 0.1% CD123+ pDCs < 0.1% 0.1% 3.6% 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

  6. Coculturewith CD4 T lymphocytes enhancesHIV-1 replication in primarypDCs N.S. *** • Downregulation of SAMHD1 in pDCcoculturedwith CD4 T lymphocytes wasassociatedwith an increased HIV-1 in coculturedpDCs. % of Infected CD123+pDCs N.S. * Median Fluorescence Intensity (MFI) in CD123+pDCs SAMHD1 • Vpx of HIV-2 and SIVmac can degrade SAMHD1 in mDCs(Laguetteet al., Nature 2011) + + + • HIV-1BaL: - - + • CD4 T-cells: - - + • VLP-Vpx: (72h PI, HIV-1BaL, n = 3 - 9 donors) (means±SEM, Two-tailed Paired t test)

  7. bNAbs CCR5 X CD4 X X X X X X Exosome Production of new virion X X Dendriticcells MVB Receptor Virological Synapse X InfectedDCs Lyse Two modes of HIV-1 transfer describedin trans and in cis CD4 T-cells Cis-infection Trans-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 ?

  8. Single cycle of T/F HIV-1Bx11 infection ? pDCs T-cells T/F HIV-1Bx11 Protease inhibitor Indinavir (IDV): Prevent final assembly and maturation of new virions * N.S. • 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. 49% cis-infection InfectedCells (% of control) 51% trans-infection + + + + Infected pDC: • To investigate the inhibition of T/F HIV-1 transfer • in trans from pDCs to CD4 T-cells by bNAbs + + + + CD4 T-cells: - - + + IDV (1 µM): In the coculture (InfectedpDC + CD4 T-cells) (72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)

  9. HIV-1 bNAb VRC01 inhibits HIV-1 transferfrom pDCsto CD4 T lymphocytes T/F HIV-1Bx11 bNAb: VRC01 Trans-infection N.S. N.S. N.S. * * N.S. * * 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 transferwith a similarefficiency as cell-free infection of CD4 T-cells InfectedCells (% of control) InfectedCells (% of control) - - - • VRC01 (µg/ml): 20 20 20 - - - • VRC01 • (µg/ml): 2 2 2 20 20 20 + + + + + + • IDV (1 µM): Infected T-cells T-cells Infected pDC Infected T-cells T-cells Infected pDC In the coculture (InfectedpDC + T-cells) Cell-to-Cell Transmission In the coculture (InfectedpDC + T-cells) Cell-to-Cell Transmission Neutralization Cell-Free Infection Neutralization Cell-Free Infection • 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 (72h PI, HIV-1Bx11, n = 4 donors) (means±SEM, Two-tailed Paired t test)

  10. HIV-1 bNAb PGT121 inhibits HIV-1 transfer from pDCs to CD4 T lymphocytes T/F HIV-1Bx11 bNAb: PGT121 Trans-infection • PGT121 alsoinhibited HIV-1 transfer and cell-free infection of CD4 T lymphocytes InfectedCells (% of control) InfectedCells (% of control) - - - • PGT121 (µg/ml): 5 5 5 - - - • PGT121 • (µg/ml): 5 5 5 + + + + + + • IDV (1 µM): Infected T-cells T-cells Infected pDC Infected T-cells T-cells Infected pDC In the coculture (InfectedpDC + T-cells) Cell-to-Cell Transmission In the coculture (InfectedpDC + T-cells) Cell-to-Cell Transmission Neutralization Cell-Free Infection Neutralization Cell-Free Infection (72h PI, HIV-1Bx11, one representative experiment was shown)

  11. Induction of pDC maturation in the presence of CD4 T lymphocytes T/F HIV-1Bx11 N.S. (1-way ANOVA, Kruskal-Wallis test) ** * % CD83+ CD123+ pDCs - - + + + + + + • HIV-1Bx11: - - - - - 20 20 2 • VRC01 (µg/ml): - - - - - - + + • IDV (1 µM): pDCs 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)

  12. Increase of IFN-α production by infectedpDCsin the presence of CD4 T lymphocytes T/F HIV-1Bx11 N.S. (1-way ANOVA, Kruskal-Wallis test) * IFN-α (% of control) IFN-αbetween 10 ~ 200 ng/ml - - + + + + + + • HIV-1Bx11: - - - - - 20 20 2 • VRC01 (µg/ml): - - - - - - + + • IDV (1 µM): pDCs pDCs + CD4 T-cells • 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 • 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 (72h PI, HIV-1Bx11, n = 3 donors) (means±SEM, Two-tailed Paired t test)

  13. Conclusion and Highlights HIV Mucosal surface (genitalmucosa) Lesion Mucous layer Submucosalepithelium • pDCs poorly support viral replication. However, in a physiologically relevant • coculture model, HIV-1 replicationincreases in pDCscoculturedwith T-cells Langerhanscell • pDCs efficiently transfer HIV-1 to adjacent CD4 T lymphocytes 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 vaccination directly at mucosal site to prevent the • early dissemination of HIV-1 after sexual transmission Virological synapse HIV infection Transmission Dissemination Exosome HIV Cell-Free Infection HIV Cell-to-Cell Transmission

  14. 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) Antibodiesobtainedfrom : John Mascola(VRC01,NIH, Bethesda, MD) Dennis Burton & Pascal Poignard (PGT121, Scripps Research Institute, La Jolla, CA)

  15. Gating strategy for detection HIV-1 replication Living CD123+ pDC CD123+ p24+ SSC SSC Live/Dead CD123+ Living CD3+ CD4 T cells SSC CD123+ FSC SAMHD1+ SSC SSC p24+ CD3+ Live/Dead SAMHD1+

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