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For Research Use Only. Not for use in diagnostic procedures. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell and Iso-Seq are trademarks of Pacific Biosciences of California, Inc. All other trademarks are the property of their respective owners. © 2015 Pacific Biosciences of California, Inc. All rights reserved.
Figure 2. (A) Phylogeny of monoclonal antibodies showing progressive divergence from unmutated common ancestor (UCA). MPI = Months Post-Infection. (B) Computational prediction of PG9-like V1/V2 apex epitope from heterologous neutralization data. (C) PG9 docked into trimer, showing region of contact.
We developed a Pacific Biosciences single molecule, real-time sequencing protocol to deeply sequence full-length env from HIV RNA, and a bioinformatics pipeline to analyze such sequences. We longitudinally examined env populations (12 time points) from a subtype A infected individual from the IAVI primary infection cohort (Protocol C) who developed bNAbs (Serum ID50>50 for 62% of a diverse panel of 105 viruses) targeting the V1/V2 region.
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V30_5743_CONS_22
V24_3403_CONS_2
V42_2121_CON
S_3
E12_161997_c054
V36_738_CONS_1
V18_61_CONS_7
V06_500_CONS_1
V36_4833_CONS_1893
V18_4
358_CONS_1
V48_6819_C
ONS_2
V36_6445_CONS_1
V09_2443_CONS_17
V09_1308_CONS_1
V24_1816_CONS_1
V48_7159
_CONS_
13
V18_1
079_CONS_3
V12_3573_CONS_1
V09_134_CONS_1
V18_29
68_CONS_1
V48_7715_CONS_1
V30_2408_CONS_9
V24_559_CONS_18
V36_4767_CONS_1
V36_5381_CONS_1
V24_3342_CONS_1
E12_162009_c030
V36_6758_CONS_1
V48_8021_CONS_1
V42_1895_CON
S_1
V48_4242_C
ONS_1V48_
7081_CONS
_5
V24_4824_CONS_28
V18_537
2_CONS
_1
V30_3976_CONS_5V36_6720_C
ONS
_1
V12_3669_CONS_1V06_3586_CONS_1
V06_5181_CONS_1
V30_5078_CONS_1V42_3619_C
ONS
_1
V18_9002_C
ONS
_1V30_6452_CONS_1
V03_4613_CONS_12
V09_1881_C
ONS
_2
V18_3552_CON
S_1
V18_3052_CONS_1
V42_5385_CONS
_5V42_1936_CONS
_1
V06_2338_CONS_8
V24_3249_CONS_1
V30_1813_CONS_10
V30_5637_CONS_2
V48_3
505_CONS_1
V09_1577_CONS_37
V30_2986_CONS_1
V18_513
6_CONS
_1
V18_30
58_CON
S_1
V42_5103_CON
S_4
V06_4944_CONS_1
V48_4546_CONS_1
V24_2801_CONS_1
V42_166_CO
NS_23
V48_7281_CONS_1
V36_535_CONS_1
V06_3320_CONS_1
V30_2957_CONS_1
V24_1614_CONS_1
V36_4691_CONS_1
V36_9041_CONS_2
V18_13
27_CO
NS_1
V30_5281_CONS_21
V48_3846_CONS_1
V36_923_CO
NS_1
V18_3601_CONS_1
V06_4369_CONS_1
V12_2589_CONS_1
V18_3489_CONS_1
V30_1031_CONS_31
V03_4717_CONS_55
V06_4645_CONS_218
V24_37
77_CON
S_18
V06_6861_CONS_1
V18_2192_CONS_1
V18_8172_C
ONS
_2
V42_5985_C
ONS
_8
V42_463_CONS_2
V48_7292
_CONS_1
V42_4337_CONS_1
V36_5712_CONS_4
V18_9198_CONS
_1
V06_3714_CONS_1
V42_756_CO
NS_21
V06_2832_CONS_1
V18_3
730_CONS_1
V06_4192_CONS_7
V24_3414_C
ONS_1
V48_4570_CONS_3V42_1384_CONS
_1
V18_5704_CON
S_1
V18_9
227_CONS_1
V03_1226_CONS_4
V48_5708_CONS_1
V24_3657_C
ONS_16
V30_3517_CONS_1V48_
3492_CONS
_1
E12_162013_c006
V06_6782_CONS_1
V48_6887_C
ONS
_5
V24_4673_CONS_4
V24_1794_CONS_4
V30_5008_CONS_69
V36_5428_CONS_2
V48_49_CON
S_3
V18_3
115_CONS_1
V36_9114_CONS_16
V48_4595_CON
S_2
V06_6184_CONS_1
V18_97
25_CO
NS_1
V18_377
9_CONS
_1V18_
3902_CO
NS_2
E12_162000_015
V42_54
74_CO
NS_5
E12_162000_c027
V42_4210_CON
S_8
V06_3846_CONS_1
V48_5627_CONS_1
V24_263_CONS_2
E12_162000_c022
V06_3927_CONS_3
V12_4447_CONS_1
V42_3039_CONS_9
V09_1572_C
ONS
_2
V18_91
96_CON
S_48
V30_3196_CONS_8
V48_3539
_CONS_1
V06_1077_CONS_1
V36_9019_CONS_1
V18_9077_CONS
_1
V06_5869_CONS_1
V36_9878_CONS_1
V06_6985_CONS_1
V06_5941_CONS_1
V48_7085_CONS_1
V09_1075_CONS_8
V06_4370_CONS_1
V18_1575
_CONS_2
V18_7679_CONS_1
V12_3082_CONS_6
V18_4462_C
ONS
_1V30_6138_CONS_4
V24_2574_CONS_499
V42_6686_C
ONS
_13
V00_21_CONS_1
V36_2490_CONS_64
V42_5663_CON
S_15
V09_1867_CONS_1
V30_6037_CONS_2
V36_186_CONS_2
V48_7743_CONS_1
V42_15
58_CO
NS_3
V18_3562_CONS_1
V24_5056_CO
NS_2
V18_5842_CONS
_1
V48_572_CONS_4
V36_4655_CONS_2
V12_307_CO
NS_1
V48_1422_C
ONS_1
V18_1728_CON
S_4
V36_3355_CONS_1
V36_1505_C
ONS_
1540
V09_35_CONS_1
V48_817
6_CONS
_1
V03_3610_CONS_1001
V06_5050_CONS_1
V18_2565_CON
S_7
V09_406_CONS_6
V36_5067_CONS_15
V48_7928_
CONS_2
V12_3698_CONS_10
V36_6951_CONS_2
V42_15
71_CO
NS_42
V30_46_CONS_7
V36_8246_CONS_10
V48_2
270_CONS_1
V18_6593_CONS_1
V36_9980_CONS_1
V06_4214_CONS_1
V18_408_CO
NS_1
V03_2692_CONS_193
V06_6854_CONS_1
V06_3169_CONS_2
V42_6151_C
ONS
_7
V06_507_CONS_49
V42_5770_C
ONS
_1
V18_6
018_CONS_1
V30_2283_CONS_5
V18_8905_CONS_31
V36_267_CONS_3
V06_7226_CONS_1V12_209_CO
NS_1
V48_377_CO
NS_3
V42_1070_CONS_2
V12_4236_CONS_1
V12_1867_C
ONS
_2
V18_4089
_CONS_1
V42_4679_CONS_4
V06_6456_CONS_1
V18_5484_CON
S_1
E12_162006_c040
V18_5292_CONS_8
V36_3243_CONS_1
V42_2566_CON
S_1
V48_1008_CONS_1
V18_2536_CONS_1
V18_9485_CONS_1
V48_2003_CONS_1
V06_1830_CONS_24
V36_1191_CONS_1
V03_2278_CONS_1
V48_8559
_CONS_1
V42_2280_CON
S_1V36_7869_CO
NS_1
V36_2489_CONS_1
V18_4376_CON
S_1
E12_162006_029
V18_7156_CONS_2
V18_6812_CON
S_1
V48_8497_CONS_17
V03_3118_CONS_3
V06_5523_CONS_19
V03_2591_CONS_1
V09_1376_CONS_3
V02_1364_CONS_12
V36_475_CONS_93
V48_8353_CONS_6
E12_161997_110
V18_35
89_CON
S_2
V18_7974_CONS_3
V30_119_CONS_12
V48_1521_C
ONS
_1
V18_8124_C
ONS
_2
V42_3035_CON
S_1
E12_162000_c032
V12_2887_CONS_1
V30_1979_CONS_12
V36_1026_CONS_42
V36_8058_C
ONS
_1
V48_2809
_CONS_4
V30_1941_CONS_2214
V42_3640_CON
S_2
V09_2221_CONS_2
V18_91
91_CONS_77
V06_377_CONS_1
V24_5140_CONS_2
V36_7612_CONS_1
V18_9066_CONS_1
V36_8931_CONS_1
V18_5029_CONS_1
V18_7681_C
ONS
_3
V42_182_CO
NS_7
V06_1961_CONS_1
V18_1792
_CONS_2
V42_3342_CON
S_1
V18_804
4_CONS
_1V18_9125
_CONS_1
V42_3286_CON
S_2
V36_9705_CONS_1
V06_3132_CONS_1
V30_881_CONS_5
V36_3580_CONS_21
V36_6792_CONS_1
V48_3771_CON
S_5
V18_1605_CONS_1
V42_1631_C
ONS
_1
V30_1145_CONS_1
V36_3661_C
ONS
_2
V42_1854_CONS
_1
V06_1242_CONS_1
V36_5577_CONS_5
E12_162006_c023
V36_7054_CONS_1V18_7046_CONS
_27
V24_1977_C
ONS_1
V24_4181_CONS_1
V06_1755_CONS_1
V48_386_CO
NS_2
V18_9567_CON
S_1
V42_4826_CONS_1
V12_2025_CONS_1
V36_7130_CONS_1
V42_3268_CONS
_1V03_9_CONS_1
V36_5509_CONS_77
V42_6252_CON
S_4
V18_7329_CON
S_1
V42_2062_CONS_7
V36_2141_CONS_113
V36_3196_CONS_1
V03_713_CONS_1
V30_1089_CONS_1
V18_406_
CONS_7
V42_30
6_CONS_1
8
V24_3155_CONS_4
V12_2797_CONS_20
V18_4748_CONS_2
V36_7930_C
ONS
_3
V09_211_CONS_1
V24_3906_CONS_3
V12_3387_CONS_3
V24_1514_CO
NS_1
V18_3436_CON
S_2
V24_4604_C
ONS_6
V42_6585_CONS
_1
V18_81
15_CON
S_1
V18_4990_C
ONS
_1
E12_162004_c016
V48_351_CONS_11
V36_10488_CONS_3
V06_1133_CONS_1
V24_3386_CONS_5
V12_2516_CONS_1
V30_1404_CONS_1
V06_2180_CONS_1
V30_3000_CONS_1
V36_8310_CONS_1
V48_6551_CONS_1
V18_46
91_CO
NS_1
V03_725_CONS_1
V18_2634_CONS_1
V18_8844_CONS_1
V24_4859_CONS_3
V30_1795_CONS_1
V18_32
9_CON
S_1
V03_438_CONS_1
V06_1633_CONS_3
V42_66
3_CONS_1
V36_842_CONS_1
V48_807_CONS_1
V06_5836_CONS_1
V36_4278_CONS_3
V48_1
330_CONS_57
V48_5
826_CONS_9
V12_2141_C
ONS
_6
V18_4577_CONS_1
V18_66
98_CO
NS_6
V36_7001_CONS_14
V42_4158_C
ONS
_1
V42_1473_CON
S_1
V48_4
681_CONS_1
V48_3
50_CONS_1
V48_7096_CO
NS_19
V06_1339_CONS_1
V09_2001_CONS_1
Full-Length env Deep Sequencing in a Donor With Broadly Neutralizing V1/V2 Antibodies Ben Murrell1, Melissa Laird2, Elise Landais3, Caroline Ignacio1, Kemal Eren1, Pham Phung4, Ellen E. Paxinos2, Sergei L. Kosakovsky Pond1, Douglas D. Richman1, Pascal Poignard3, Davey M. Smith1 1University of California San Diego, La Jolla, CA, United States; 2 Pacific Biosciences, Menlo Park, CA, United States. 3The International AIDS Vaccine Initiative Neutralizing Antibody Center, La Jolla, CA, United States; 4Monogram Biosciences, LabCorp, South San Francisco, CA, United States.
Understanding the co-evolution of HIV populations and broadly neutralizing antibody (bNAb) lineages may inform vaccine design. Novel long-read, next-generation sequencing methods allow, for the first time, full-length deep sequencing of HIV env populations.
Introduction V2 Epitope Kinetics
Objective
Ben Murrell, Ph.D. University of California, San Diego
9500 Gilman Drive, MC0679 San Diego, CA 92093
E-mail: [email protected]
Methods HIV-1 env Phylogeny
Figure 3. Phylogeny of HIV env SMRT sequences collected from PC64 at 12 visits, compared to previously generated clonal sequences (grey boxes). Sequences with >99% similarity were collapsed, and such clusters are represented by black circles.
Conclusions
HIV-1 env Longitudinal Selection Pressure Antibodies
00 02 03 06 09 12
18 24 30 36 42 48
MPI MPI
Cluster Size 3000 2000 1000
Clonal Sequences
100
10 2 10 1 10 0 10 -1 10 2 10 1 10 0 10 -1
80
60
40
20
0
-20
mAb concentration (ug/mL)
% N
eutra
lizat
ion
MRCA WT MRCA R166S V6_C022 WT V6_C022 S166R
MRCA WT MRCA R169II V12_C018 WT V12_C018 I169R
Extract viral RNA
cDNA Generation
PCR screen
Iterative PCR
SMRTbell™ Preparation
Align Primers/Bind Polymerase
SMRT® Sequencing
Full-length Envelope Analysis (FLEA)
Days%1'5%
Day%6%
Figure 1. Streamlined end-to-end workflow for the isolation, amplification, preparation, sequencing and analysis of full-length HIV env amplicons.
0.04
V36-10496-A9_42H1_BB
V24-11444-O12_66H0_BB
V24-11441-F08_64H0_BB
V36-10526_O08_28H0_TH
V36-10481_P08_23H0_THV36-10463-K13_39H0_TH
V12-11691-E14_45H1_BB
V36-10515-K05_33H0_BB
V36-10552_F12_29H0_TH
V36-10540-P03_44H0_BB
V18-11483-P16_53H1_BBV12-11691-K17_46H0_BB
V18-11492-F18_55H0_BB
V36-10491_B19_34H0_TH
V36-10512-J15_S182_Minority_HC
V36-10588-O08_32H0_BB
V36-10482_F16_35H0_TH
V36-10559_N23_30H0_TH
V12-11691-N23_47H1_BB
V18-11522-I02_58H0_BBV18-11504-F11_56H0_BB
V36-10521_O05_26H0_TH
V36-10502_I02_24H0_TH
V24-11424-D18_59H0_BB
V36-10519_H05_38H0_TH
V24-11424-M03_61H0_BB
V12-11697-A10_49H0_BBV24-11428-H17_62H0_BB
V36-10559-N23_30H0_BB
V36-10515_K05_33H0_TH
V36-10588_O08_32H0_THV36-10588-O8_S221_clust3_546_0.992565
V18-11491-O04_54H1_BB
V24-11441-I20_65H0_BB
PC64_VHGL_BBds
V12-11697-D15_50H0_BB
V12-11694-G14_48H1_BB
V36-10540-P3_S196_clust1_524_0.98665
V18-11505-H16_57H0_BB
V36-10467-L22_21H0_TH
V36-10488-L17_41H0_BB
V36-10532_D06_27H0_TH
V12-11702-C19_52H0_BB
V36-10488_L17_25H0_TH
V36-10505-H04_43H0_BB
V24-11424-I09_60H0_BB
V24-11438-N14_63H0_BB
V12-11700-N17_51H0_BB
UCA 12 18 24 36
MPI PG9[1]
gp120
gp41
Env
HC
LC
Details: • Predicted from IC50s against 105 heterologous viruses • Novel machine learning algorithm • MCMC on P(IC50|Sequences)
Computational Epitope
Prediction PC64101(21H0):
PG9-like V1/V2 Apex
(A)
(B)
(C)
Functional Characterization
(A) 00-02 MPI 02-03 MPI 03-06 MPI 06-09 MPI
09-12 MPI 12-18 MPI 18-24 MPI 24-30 MPI
30-36 MPI 36-42 MPI 42-48 MPI Combined Positive
Purifying
Positive
Purifying
Figure 5. Figure 6. (A) Neutralization of PC64 autologous pseudoviruses by a PC64V36 mAb (B) Color-coded decrease in neutralization IC50 for single aa JRCSF mutant pseudovirus (AlaScan) compared to wildtype by PC64V36 mAbs, displayed on trimer structure[2].
(A) (B)
Figure 6. Selection at sites and over time. For each pair of contiguous samples, strength of selection was inferred using FUBAR[3], plotted from purifying selection (blue) to positive selection (red). As early as 3 to 6 MPI, strong positive selection can be seen at the trimer apex.
• Longitudinal full-length HIV env deep sequencing allows:
• Accurate phylogenetic inference
• Detailed view of epitope escape dynamics
• Synthesis of observed Env proteins for functional characterization
• In PC64, concurrent epitope diversity (3-12 MPI) immediately preceded the development of heterologous serum activity. Waves of sequential escape preceded rapid breadth increases.
160
181
166 167
169
Epitope Positions
References
ACKNOWLEDGEMENTS: This research was supported in part by the National Institutes of Health (AI090970, AI100665, AI036214, U01GM110749, GM093939), and the UCSD Center for AIDS Research (Developmental Grant, AI36214, Bioinformatics and Information Technologies Core)
[1] McLellan, J.S., et al. (2013). Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature. [2] Pancera, M., et al. (2014)., Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature. [3] Murrell, B. et al. (2013). FUBAR: A Fast, Unconstrained Bayesian AppRoximation for inferring selection. Molecular Biology
and Evolution.
Figure 4. (A) V2/apex epitope configurations, comprising residues of interest for the primary antibody lineage. Colors serve as a key for the frequency curves in (B), where concurrent epitope diversity (3-12 MPI) is followed by sequential selective sweeps. (C) Serum breadth and viral load over time, showing initial development of heterologous neutralization after a period of dramatic concurrent diversity at the apex, followed by increases in breadth that may be a response to viral selective sweeps.
NRNRI!
181 169 167 166 160 HXB2#:
AA:
NRNRL! NRNIL!
NRDRL!
NSNRL! NSNRI!
NRDIL! NKDIL!
DRDIL!
NSDKL!
NKDTL!
NKDEL!
(B)
Freq
uenc
y
0 10 20 30 400.00.20.40.60.81.0
MPI
Frequency
160 & 166 & 167 & 169 & 181 NRNRI
NRNRL
NSNRI
NRDRL
NSNRL
NRNIL
NRDIL
NKDIL
NSDKL
DRDIL
NKDEL
NKDTL
0 6 12 18 24 30 36 42 48
1.0
0.8
0.6
0.4
0.2
0.0
Seru
m B
read
th
100
1000
10000
100000
1000000
0
0.5
1
1.5
2
2.5
3
0 6 12 18 24 30 36 42 48
3.0
2.5
2.0
1.5
1.0
0.5
0.0 0 6 12 18 24 30 36 42 48
Vira
l Loa
d
106
105
104
103
102
(C)
Months Post-Infection
Months Post-Infection
Colors match curves
