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

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

Andrabi R et al PLoS one , 2012 (in press)AIDS Vaccine 2011, Bangkok Thailand

Screening and epitope characterization of cross neutralizing plasma antibodies

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AIIMS206AIIMS239AIIMS249A1A2

gp120 gp41

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C2

V3

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-V5 C

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Overlapping peptides

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Land A et al., Biochimie. 2001 Aug;83(8):783-90

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Plasma samples

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Immunogenic regions

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p=0.3413

Drug status

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Andrabi R et al Arch. Virol. 2011 Oct;156(10):1787–94

Immunodominance of V3 over MPER; Association with HIV viremia

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RNA copies/ml plasma (Viral Load)

MPER (r=-0.127 p=0.373)V3 (r=0.286 p=0.037)*

MPER

V3

Log. (MPER)

Log. (V3)

V3 loop MPER

0 1000 2000 30000tan28a566028

0tan19a566019

0tan9a56609

0tan29a566029

0tan19a566019

0tan10a566010

n=80 (r=0.29 p=0.008)

Days from first diagnosis

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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)

V1/V2

V3

CD4 bs

B.S.

CD4ChemokineReceptor

gp120

by J. Hoxie

gp120/CD4/Co-receptor interactionsSequence of events

Features of third variable region

• The third variable (V3) is divided intobase, 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

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 patientsa) Antiretroviral drug naïveb) Infected for more than 3 years orc) Plasma positive for neutralization

Antibodies were selected with Cholera Toxin-Bprotein containing con-C V3 (V3C-CTB)

V3-CTB bound to 447-52D fab fragment

Totrov M et al Virology 405 (2010) 513–523

Antibody isolation and characteristics; summary

Number of HIV-1 infected patients 33

Total number of PBMCs isolated (million) 321.2

Number of wells plated (96 well plate) 3321

Number of wells positive for V3-CTB 199

Number of samples reaching hybridoma fusion stage 25

Number of samples reaching cloning stage 15

Number of anti-V3 antibody secreting cell lines 3

# mAb Specificity Isotype IGHV CDR H3 1 277 V3 (gp120) IgG1 κ 3-30*03 ATLPVVTPATEPFDFW 2 903 V3 (gp120) IgG1 κ 3-30*03 AKHYAEGGLDVW 3 904 V3 (gp120) IgG1 λ 1-8*01 ARFALQSYIVSTDSYDIDYW

Andrabi R et al Virology Journal, 2012 (In press)

Binding of anti-V3 mAbs to peptides and proteins

Relative affinity of anti-V3 mAbs to peptides and proteins

anti-V3 mAbsProtein/peptide Subtype 277 903 904

447-52D

1418

Con-C V3 C 0.33 0.109 0.046 1.2 >10Con-B V3 B >10 >10 0.623 0.323 >10Du156.12 C 0.047 0.019 0.009 0.035 >10JRFL B >10 >10 0.04 0.01 >1092RW020 A 0.098 0.05 0.028 0.323 >10SF162 B >10 >10 >10 0.004 >10MN B >10 >10 2.27 0.011 >10MPER C >10 >10 >10 >10 >10IDR C >10 >10 >10 >10 >10p24 B >10 >10 >10 >10 >10BSA NA >10 >10 >10 >10 >10PP NA >10 >10 >10 >10 >10

Amino acid sequence of C2-V3-C3 overlapping peptides anti-V3 mAbs

Peptide ID IIVHLNESVEIVCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEEKWNKTLQ 277 903 904 447-52D 1418

9256 IIVHLNESVEIVCTR >10 >10 >10 >10 >109257 LNESVEIVCTRPNNN >10 >10 >10 >10 >109258 VEIVCTRPNNNTRKS >10 >10 >10 >10 >109259 CTRPNNNTRKSIRIG >10 >10 >10 >10 >109260 NNNTRKSIRIGPGQT >10 >10 >10 0.049 >109261 RKSIRIGPGQTFYAT 4.04 1.37 0.327 0.149 >109262 RIGPGQTFYATGDII >10 >10 >10 >10 >109263 GQTFYATGDIIGDIR >10 >10 >10 >10 >109264 YATGDIIGDIRQAHC >10 >10 >10 >10 >109265 DIIGDIRQAHCNISE >10 >10 >10 >10 >109266 DIRQAHCNISEEKWN >10 >10 >10 >10 >109267 AHCNISEEKWNKTLQ >10 >10 >10 >10 >10

Epitope mapping of anti-V3 mAbs with consensus-C V3 overlapping peptides

Andrabi R et al Virology Journal, 2012 (In press)

Neutralization of HIV viruses by anti-V3 mAbs in TZM-bl assay

anti-V3 mAbs

# Virus ID Tier Clade 277 903 904 447-52D 14181 MW965.26 1A C 0.34 0.15 <0.1 <0.1 >302 DJ263.8 1B A 8.2 1.7 0.4 <0.1 >303 SF162.LS 1A B >30 >30 >30 <0.1 >304 92RW009 - A >30 >30 >30 >30 >305 JR-CSF - B >30 >30 >30 0.3 >306 HIV-001428 2 C >30 15.4 22.8 >30 >307 ZM109F.PB4 2 C >30 >30 >30 >30 >308 ZM233M.PB6 2 C >30 >30 >30 >30 >309 Du156.12 2 C >30 >30 >30 >30 >3010 JRFL 2 B >30 >30 >30 20 >3011 RHPA4259.7 2 B >30 >30 >30 >30 >30

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)

Exposure of epitopes for anti-V3 mAbs

Andrabi R et al Virology Journal, 2012 (In press)

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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

Acknowledgements

Funding

AIIMSKalpana LuthraSubrata SinhaRajesh KumarAmbili NairPatik KumarNaveet WigAshutosh Biswas

Safdarjung HospitalManju Bala

National Institute of ImmunologyRahul Pal

NYU School of MedicineSusan Zolla PaznerMiroslaw K GornySuman LalConstance Williams

All the study participants

Fogarty center for AITRPNIH, ARRRP