1. Production and characterization of human anti-V3 monoclonal antibodies from Indian clade C HIV-1 infected patients Raiees Andrabi 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. Andrabi R et al PLoS one, 2012 (in press) AIDS Vaccine 2011, Bangkok Thailand Screening and epitope characterization of cross neutralizing plasma antibodies Land A et al., Biochimie. 2001 Aug;83(8):783-90

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

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

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

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 a)Antiretroviral drug naïve b)Infected for more than 3 years or c)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 Number of HIV-1 infected patients33 Total number of PBMCs isolated (million)321.2 Number of wells plated (96 well plate)3321 Number of wells positive for V3-CTB199 Number of samples reaching hybridoma fusion stage25 Number of samples reaching cloning stage15 Number of anti-V3 antibody secreting cell lines3 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 anti-V3 mAbs Protein/pep tide Subtype277903904 447- 52D 1418 Con-C V3 C0.330.1090.0461.2>10 Con-B V3 B>10 0.6230.323>10 Du156.12 C0.0470.0190.0090.035>10 JRFL B>10 0.040.01>10 92RW020 A0.0980.050.0280.323>10 SF162 B>10 0.004>10 MN B>10 2.270.011>10 MPER C>10 IDR C>10 p24 B>10 BSA NA>10 PPNA>10 Amino acid sequence of C2-V3-C3 overlapping peptidesanti-V3 mAbs Peptide ID IIVHLNESVEIVCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEEKWNKTLQ 277903904447-52D1418 9256 IIVHLNESVEIVCTR >10 9257 LNESVEIVCTRPNNN >10 9258 VEIVCTRPNNNTRKS >10 9259 CTRPNNNTRKSIRIG >10 9260 NNNTRKSIRIGPGQT >10 0.049>10 9261 RKSIRIGPGQTFYAT 4.041.370.3270.149>10 9262 RIGPGQTFYATGDII >10 9263 GQTFYATGDIIGDIR >10 9264 YATGDIIGDIRQAHC >10 9265 DIIGDIRQAHCNISE >10 9266 DIRQAHCNISEEKWN >10 9267 AHCNISEEKWNKTLQ >10 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 IC 50 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: IC 50 1 µg/ml (yellow) Andrabi R et al Virology Journal, 2012 (In press)

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

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 Funding 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 All the study participants Fogarty center for AITRP NIH, ARRRP