Mechanisms of HIV-1 Resistance to ADCC David Evans University of Wisconsin-Madison July 26, 2017 “No conflicts of interest to declare”

Perspective HIV-1 has evolved to replicate continuously in the presence of active host immune responses, and has therefore acquired mechanisms to overcome all immune responses in most infected individuals. The ability of neutralizing antibodies to block HIV-1 infectivity is dependent on their capacity to bind to native conformations of the viral envelope glycoprotein as they exist on the surface of virions.

ADCC Assay Antibody Env CD16 HIV-1 Tat NK Cell No plasma No virus LTR Luc. 0% 100% Serial antibody dilutions No virus No plasma Similarity to assays for neutralizing antibodies. Alpert et al. J. Virol. 2012

Env Expression on the Surface of HIV-infected is Tightly Regulated Gardiner et al. J. Virol. 2016

Env Internalization Protects HIV-infected Cells from ADCC HIV-1 gp41 tail 710 WT: NRVRQGYSPL SFQTHLPIPR Y710G: ......G... .......... Y710F: ......F... .......... % RLU % MAX Env HIVIG (µg/ml) von Bredow et al. J. Virol. 2015

Tetherin Adapted from Gottlinger. 2008. Nature

Vpu Downregulates and Degrades Tetherin bTrCP-2 Vpu Lysosomal Degradation

Neil et al. 2008. Nature

Vpu Protects HIV-infected Cells from ADCC

RNAi Knockdown of Tetherin, but not CD4 or NTB-A, Increases Resistance to ADCC Tetherin CD4 NTB-A Arias et al. PNAS. 2014

CD4 Downmodulation by Nef and Vpu Ab CD4 Env CD4 Env Nef Vpu Downregulation & Degradation

CD4 Downmodulation Prevents the Exposure of CD4-Inducible Epitopes

Sensitivity of Wildtype versus Nef- and Vpu-Deleted HIV-1 CH77 T/F to Plasma from HIV+ Donors

HIV-1 Env Trimer Glycan Shield CD4 Binding Site (CD4bs) V3 gp120 CD4bs Viral Membrane or Infected Cell Membrane Glycan Shield CD4 Binding Site (CD4bs) gp120 gp41 V3 CD4bs MPER Burton et al. Cell Host & Microbe. 2012

Broadly Neutralizing Antibodies % RLU von Bredow et al. J. Virol. 2016 mAb (µg/ml)

Non-Neutralizing Antibodies % RLU von Bredow et al. J. Virol. 2016 mAb (µg/ml)

ADCC Correlates with Neutralization

Conclusions There is little Env on the surface of HIV-infected cells due to trafficking signals in cytoplasmic tail of gp41. Tetherin antagonism by Vpu protects HIV-infected cells from ADCC by preventing the accumulation of captured virions on the cell surface. CD4 downmodulation by Nef and Vpu protects HIV-infected cells from ADCC by preventing the exposure of CD4-inducible epitopes in Env. Many of the same structural features of the Env trimer that afford resistance to neutralizing antibodies also afford resistance to ADCC.

Acknowledgements University of Wisconsin University of Montreal Benjamin von Bredow Andres Finzi Juan Fernando Arias Lisa Heyer Scripps Research Institute Michael Alpert Dennis Burton Jaye Gardiner New York University Nathan Sherer Susan Zolla-Pazner Tulane University James Robinson AI121135

CD4 Downmodulation by Vpu and Nef Prevents the Exposure of CD4-Inducible Epitopes on the Surface HIV-infected Cells Veillette et al. J. Virol. 2014

CD4 Downmodulation by Vpu and Nef Protects HIV-infected Cells from ADCC Veillette et al. J. Virol. 2015

ADCC Correlates with Binding to Env on the Surface of Infected Cells and with Virus Neutralization von Bredow et al. J. Virol. 2016

Tetherin/BST-2/CD317 Klimkait & Orenstein. 1990. J. Virol. Kupzig et al. 2003. Traffic Tetherin, also known as BST-2 or CD317, was identified a little over 3 years ago by Stuart Neil and Paul Bieniasz, and independently by Nicole Van Damme and John Guatelli, as the interferon-inducible host-cell protein that interferes with the detachment of vpu-deleted HIV-1 from certain restrictive cells. This factor accounts for the late stage defect in the release of virus particles shown here by electron microscopy. In the absence of Vpu, the virus can complete assembly and budding, note that the viral envelope of these particles is discontiguous with the plasma membrane of the cell. However, the virus remains attached to the surface of the cell. The explanation for this phenotype was provided with the identification of tetherin. This molecule has a rather unusual topology that includes a an N-terminal cytoplasmic domain, a membrane-spanning domain, an extracellular coiled-coil domain and a site for the addition of a glycosyl phosphatidyl inositol tail at the C-terminus. Based on these structural features that enable both ends of the molecule to be anchored in lipid membranes it was proposed that… interferon-inducible transmembrane protein that interferes with the release of vpu-deleted HIV-1 from restrictive cells Neil et al. 2008. Nature Van Damme et al. 2008. Cell Host & Microbe

IFNa Treatment of HIV-infected Cells Increases their Susceptibility to ADCC Arias et al. PNAS. 2014

Vpu Substitutions Differentiate Tetherin Antagonism from CD4- and NTB-downmodulation Tetherin Vpu A14L X S52N S56N X bTrCP Sequestration Degradation A14L – blocks binding to tetherin, but does not affect CD4 I46K – blocks CD4 binding S52N, – prevents tetherin degradation, S56N but not tetherin sequestration – does not affect NTB-A Arias et al. PNAS. 2014

CD4 Binding Exposes Surfaces of the Inner Domain of gp120 Recognized by Non-neutralizing Antibodies Veillette et al. J. Virol. 2014

Exceptions…Gp41 MPER-Specific bNAbs and 2G12 % RLU mAb (µg/ml)

Sensitivity of Wildtype versus Nef- and Vpu-Deleted HIV-1 CH77 T/F to Monoclonal Antibodies

Mechanisms of HIV-1 Resistance to ADCC Env internalization Tetherin antagonism CD4 downmodulation Structural features of the Env trimer

Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) Env SIV or HIV infected cell NK cell CD16 Granules Perforin Granzyme

Acknowledgements University of Wisconsin University of Montreal Benjamin von Bredow Andres Finzi Juan Fernando Arias Lisa Heyer Scripps Research Institute Michael Alpert Dennis Burton Jaye Gardiner New York University Nathan Sherer Susan Zolla-Pazner Tulane University James Robinson AI121135