Download - Raiees Andrabi AIIMS, New Delhi, India
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
100
1000
10000
AIIMS206AIIMS239AIIMS249A1A2
gp120 gp41
V2
C2
V3
C4
-V5 C
5-F
P
IDR
CH
RC
HR
-MP
ER
MP
ER
CT
Overlapping peptides
Rec
ipro
cal M
ax50
Bin
din
g T
iter
Land A et al., Biochimie. 2001 Aug;83(8):783-90
1000
10000
100000 V3 loop
Max
50 b
ind
ing
tite
r
1000
10000
100000
MPER region
Plasma samples
Max
50 b
ind
ing
tite
r
V3
MPER
1000
10000
100000 p<0.0001 ***
Immunogenic regions
Rec
ipro
cal M
ax50
Bin
din
g T
iter
V3 Nai
ve
V3 Tre
ated
MPER N
aive
MPER T
reat
ed
1000
10000
100000
p<0.0001
p<0.0001
p=0.3413
Drug status
***
p<0.0001
***
***
Rec
ipro
cal
Max
50 B
ind
ing
Tit
er
Andrabi R et al Arch. Virol. 2011 Oct;156(10):1787–94
Immunodominance of V3 over MPER; Association with HIV viremia
118
118 (
1)118 (
2)
149
149 (
1)149 (
2)149 (
3)
151
151 (
1)151 (
2)
154
154 (
1)154 (
2)1000
10000
100000
A
Sample ID
Rec
ipro
cal M
ax50
Bin
din
g T
iter
118
118 (
1)118 (
2)
149
149 (
1)149 (
2)149 (
3)
151
151 (
1)151 (
2)
154
154 (
1)154 (
2)
1000
10000
100000
B
Sample ID
100
1000
10000
100000
10 100 1000 10000 100000 1000000 10000000Reci
proc
al M
ax50
Bin
ding
Tit
er
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
% c
ros
s r
ea
cti
ve
an
ti-V
3
an
tib
od
ies
p=0.0001
1000
10000
100000Con-CCon-B
Plasma samples
Rec
ipro
cal
Max
50 B
ind
ing
Tit
er
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)
0
100
200
300
400 Du156.12_VirusJRFL_Virus
Vir
us
cap
ture
d(p
24 p
g/m
l)
277
903
904
447
1418
0
20
40
60
80
100 Du156.12_VirusJRFL_Virus
Monoclonal antibodies (mAbs)
% N
eutr
aliz
atio
n 447-52D and SF162
277
903
904
447
1418
0
10
20
30
100
200
300
1000
2000
3000 Q461_AJRFL_BRHPA4259.7_BDu156.12_CZM53M.PB12_CAIIMS201_CAIIMS254_CAIIMS261_CAIIMS212_C
Monoclonal antibodies (10g/ml)
Vir
us
cap
ture
d (p
24 p
g/m
l) Intact virion binding
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