Neutrophils mediate potent and rapid anti-HIV antibody-dependent functions Matthew J Worley, Anthony D Kelleher, Stephen J Kent and Amy W Chung The University of Melbourne, Australia

FcR mediated antibody functions Antibodies can recruit innate cells via FcR NK cells can mediate ADCC responses via an Fcγ receptor Monocytes and neutrophils are able to mediate phagocytosis and ADCC responses via Fcγ receptors

FcR mediated antibody functions Virus Phagosome Antibody Granules Kill Fc Receptor Antigen Antibody Infected cell Fc Receptor Effector cell

FcR mediated antibody functions in HIV High levels of ADCC antibodies correlate with delayed disease progression with HIV (Baum et al. 1996, Chung et al. 2011, Wren et al. 2013) The RV144 vaccine trial highlighted the importance of Fc mediated antibody functions IgG induced ADCC to envelope protein (Haynes et al. 2012) IgG3 to the V1V2 region reduced risk of infection (Yates et al. 2014) IgG3 mediated polyfunctional immune responses (Chung et al. 2014) NK cells and monocytes have been the main focus of recent studies

Leukocyte cell prevalence FcγRI FcγRIIIB FcγRIIB FcγRIIB FcγRIIA FcγRIIIA FcγRI Neutrophils ~65% T cells ~20% NK cells ~5% B cells ~5% FcγRIIB Monocytes ~5% FcγRIIIA FcγRIIA

Aim Cohort clinical data Can neutrophils mediate antibody-dependent anti-HIV responses? Cohort clinical data   HIV Number of samples 33 CD4 count entry, cells/μl (median, Range) 566 (330-1156) Viral load RNA copies/ml (median, Range) 26400 (382-339000)

Neutrophils are able to mediate anti–HIV antibody-dependent phagocytosis gp120 protein Fluorescent bead Antibody Fc Receptor Neutrophil Phagosome 25ug/ml IgG Method adapted from Ackerman et al. 2011

Neutrophils are able to mediate anti–HIV ADCC responses gp120 protein Lysis Antibody CFSE viability stain Fc Receptor Pkh stained membrane Loss of CFSE viability stain 25ug/ml IgG Method adapted from Gómez-Románet al. 2006

The ADNP and ADCC responses correlate 25ug/ml IgG

Leukocyte cell prevalence FcγRIIB FcγRIIIA FcγRI FcγRIIA FcγRIIB B cells ~5% FcγRI FcγRIIIB NK cells ~5% Neutrophils ~65% Monocytes ~5% FcγRIIIA T cells ~20% FcγRIIB FcγRIIA

How do neutrophil ADCC responses compare to other innate cells?

PBMC’s mediate enhanced levels of ADCC compared to NK cells at all antibody concentrations

Neutrophils perform similar levels of ADCC to PBMC’s

Monocytes perform similar levels of ADCC responses to Neutrophils and PBMC’s

How do neutrophil ADCC responses compare to other innate cells?

PBMC’s mediate enhanced levels of ADCC compared to NK cells

Monocytes mediate enhanced levels of ADCC compared to PBMC’s and NK cells

Neutrophils mediate rapid ADCC responses to HIV envelope protein

Summary and conclusions Neutrophils can mediate HIV-specific antibody-dependent phagocytosis and ADCC responses Neutrophils mediate ADCC responses faster than other innate effector cells Rapid action and high magnitude of ADCC by neutrophils highlights their potential importance early in HIV infections

Future directions Recruit a new cohort of a progressors and controllers Do neutrophils utilise other antibody-dependent mechanisms to control HIV? Do the RV144 vaccinees mediate antibody dependent functions with neutrophils?

Acknowledgment Peter Doherty Institute/ University of Melbourne Amy Chung Stephen Kent Ivan Stratov Matthew Parsons Adam Wheatley Vijaya Madhavi Hyon-Xhi Tan Sarah Lloyd Fernanda Ana-Sosa Batiz Kevin John Selva Wen Shi Lee Hillary Vanderven Joshua Glass Anne Kristensen Vinca Alcantara Thakshila Amarasena Kirby Institute/ University of New South Wales Anthony Kelleher Ansari Shaik Flow cytometry Tina Luke Members of the flow core Funding National Health and Medical Research Council