Raiees Andrabi AIIMS, New Delhi, India

1. Production and characterization of human anti-V3 monoclonal antibodies from Indian clade C HIV-1 infected patients RaieesAndrabi AIIMS, New Delhi, India AIDS 2012, Washington DC, USA

2. Rationale for the study • Subtype-C viruses cause >50% of HIV-1 infections worldwide • Limited information on antibody response in subtype-C Indian patients • Very few of mAbs have been raised from subtype-C infected individuals

3. Screening and epitope characterization of cross neutralizing plasma antibodies Land A et al., Biochimie. 2001 Aug;83(8):783-90 Andrabi R et al PLoS one , 2012 (in press) AIDS Vaccine 2011, Bangkok Thailand

4. Immunodominance of V3 over MPER; Association with HIV viremia V3 loop MPER Andrabi R et al Arch. Virol. 2011 Oct;156(10):1787–94

5. Cross reactive anti-V3 Abs and amino acid conservation of V3 loop in patient viruses p=0.0001 Andrabi R et al. BMC Infectious Diseases 2012 12(Suppl 1):P24. Andrabi R et al, J.Microbiol, 2012 (In press)

6. Sequence of events gp120/CD4/Co-receptor interactions by J. Hoxie

7. Features of third variable region • The third variable (V3) is divided into • base, stem and tip. • V3 is invariant in length and conserved in amino acid sequence. • V3 crown possesses structurally conserved elements Chih-chin Huang, et al. 11 NOVEMBER 2005 VOL 310 SCIENCE Zolla-Pazner and Cardozo, Nat. Rev. Immunol., 2010 Jiang et al., Nature Struct. Mol. Biol., 2010

8. Isolation of anti-V3 mAbs from HIV-1 infected patients • V3 is highly antigenic and V3-specific Abs display both neutralizing and ADCC anti-viral activities • V3 with GPGQ induces more potent and cross-reactive antibodies than GPGR (Gorny MK et al J Virol. 2006 Jul;80(14):6865-72) 33 HIV-1 infected patients were recruited for anti-V3 antibody production. Criteria for selection of patients • Antiretroviral drug naïve • Infected for more than 3 years or • Plasma positive for neutralization • Antibodies were selected with Cholera Toxin-B protein containing con-C V3 (V3C-CTB) V3-CTB bound to 447-52D fab fragment Totrov M et al Virology 405 (2010) 513–523

9. Antibody isolation and characteristics; summary Andrabi R et al Virology Journal, 2012 (In press)

10. Binding of anti-V3 mAbs to peptides and proteins Relative affinity of anti-V3 mAbs to peptides and proteins Epitope mapping of anti-V3 mAbs with consensus-C V3 overlapping peptides Andrabi R et al Virology Journal, 2012 (In press)

11. Neutralization of HIV viruses by anti-V3 mAbs in TZM-bl assay The IC50 values which indicate the amount of mAbs (µg/ml) needed for 50% neutralization were estimated from the titration curves and are in color-coded scale: IC50<0.1 µg/ml (red), IC50<0.1-1 µg/ml (orange), IC50>1 µg/ml (yellow) Andrabi R et al Virology Journal, 2012 (In press)

12. Exposure of epitopes for anti-V3 mAbs 447-52D and SF162 Intact virion binding Andrabi R et al Virology Journal, 2012 (In press)

13. Conclusions • The CNP mapped to ten different specificities of envelope glycoprotein • V3 region was highly immunodominant compared to MPER • The V3 sequence of the patient viruses was conserved and the plasma displayed cross-reactivity • The anti-V3 mAbs displayed cross-clade binding and neutralization potential • The epitopes recognized by anti-V3 mAbs are well exposed on intact virions • The mAbs could be tested for other antibody mediated activities

14. Acknowledgements All the study participants AIIMS Kalpana Luthra Subrata Sinha Rajesh Kumar Ambili Nair Patik Kumar Naveet Wig Ashutosh Biswas Safdarjung Hospital Manju Bala National Institute of Immunology Rahul Pal NYU School of Medicine Susan Zolla Pazner Miroslaw K Gorny Suman Lal Constance Williams Fogarty center for AITRP NIH, ARRRP Funding