1. RV305 Linear Epitope Mapping
Updated 12-Jan-2016
Ryan Duffy/ Shaunna Shen
Georgia Tomaras Laboratory
Duke Human Vaccine Institute
CA-VIMC Binding Antibody Core

2. Contents Slides 3-4: Methods Slide 5: Linear Epitope Definitions
Slide 6: Results Summary
Slide 7: Discussion
Slide 8: Table- binding magnitude summary
Slide 9: Table- percent of responses summery
Slides 10: Bar graph- positive responder rate per group
Slide 11-13: Plots- binding magnitude by group
Slides 14: Pie charts- relative percentages of specificity across gp120
Slide 15: Table and Plot- summary of anti-gD binding magnitude
Slide 16: Bar graph- binding to gD peptides by each group
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

3. Methods Sample Information: Assay Information:
Visit 5 plasma from 70 IDs were tested; corresponding RV144 v1 prebleeds were tested to define baseline binding for every PubIDs.
3 Vaccine Groups
1) ALVAC + AIDSVAX B/E
2) AIDSVAX x2
3) ALVAC x2
Assay Information:
Samples were tested at a 1:50 dilution
Blocking Solution: BAMA Diluent
Secondary Antibody: Goat Anti-Hu IgG*AlexaFluor647 at 1:500
Slide Lot: JPT2354, 2348
Scanned using PMT580, 100% Laser Power
Peptide Library: 15-mers overlapping by 12 covering:
HIV-1 gp160 of 7 Consensus Sequences (Clade A, B, C, D, Group M, CRF1, and CRF2)
HIV-1 gp120 of 6 Vaccine Strains (1.A244, 1.TH023, B.MN, C.1086, C.TV1, C.ZM651)
18 overlapping peptides covering glycoprotein D (gD) of Herpes Simplex Virus
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

4. Methods
Analysis
Baseline Subtraction: prebleed binding values (RV144 vst1) + 3 x Stdev, or 200 at least
Positivity Cutoff was determined for each peptide by using the mean visit 1 value from all 70 PTIDs + 3 x Stdev.
All binding values under cutoff were changed to 1
Neutralization data were generated by Dr. Montefiori’s Lab
Correlation with neutralization analysis: Maximum binding intensity in identified epitopes was analyzed for correlation with ID50 values of plasma neutralization against 4 tier-1 HIV-1 strains
MN.3, TH023.6, C3347.c11.LucR, and C1080.c03.LucR
Statistical analyses:
Two-tailed Spearman correlation test for correlation analysis
Two-tailed Mann Whitney U-test for magnitude comparison between groups
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

5. Linear Epitopes Definition
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Peptide Region
23-26
33-37
39-40 53
83-85
95-106
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

6. Results
Groups 1 and 2 showed similar levels of total binding, Group 3 total binding was significantly lower
Dominant responses were against V3 and C5.2
Other linear epitopes targeted include C1, V2, C2, and C4
No binding to gp41 was detected
46/70 IDs showed positive IgG response to the V2 epitope that was identified in RV144
Binding magnitude to multiple linear epitopes (including gD peptide) correlated with neutralizing activity; most significant correlation was seen with V3 binding
Anti-gD binding was observed in 57/70 IDs, with Groups 1 and 2 showing increased positivity rate and binding magnitude compared to Group 3
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

7. Discussion
We profiled linear epitopes for 70 RV305 v5 plasma samples, using corresponding v1 samples from RV144 v1 to define pre-vaccination binding. We did not map binding at RV305 entry visit (RV305 v2). Therefore the binding antibody responses measured at V5 could be a combination of:
remaining response from RV144 vaccination, or
boosted response from RV305 boosts
Multiple binding specificities correlated with neutralization. This can be explained by:
The neutralizing activity involving polyclonal mix of antibodies; or
Higher levels of vaccine induced antibody responses are indicative of better neutralization and another epitope specificity (not measured here) could be responsible for the neutralization.
- Future peptide blocking neutralization study would help answer this question.

8. Group I: ALVAC + AIDSVAX B/E
PubID
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Group I: ALVAC + AIDSVAX B/E
305-01 1
2,625
4,919
13,323
2,305
305-04
1,817
7,442
1,048
52,533
17,859
305-05
1,368
725
6,102
22,232
849
7,638
305-08
3,304
3,428
3,565
6,822
2,995
17,325
6,307
305-10
2,160
16,687
2,929
305-13
1,586
1,970
6,968
1,306
7,637
30,963
5,129
305-14
11,922
8,259
4,733
440
61,439
2,249
59,616
305-16P
305-19P
305-25
615
3,084
4,156
23,554
3,406
2,458
305-27
684
4,410
6,639
2,209
29,701
35,029
932
42,577
305-28
254
480
4,940
20,267
4,042
305-30
409
1,184
646
281
7,426
1,704
305-34
1,677
3,648
21,539
3,934
1,281
305-42
14,238
6,634
186
42,722
412
24,470
305-44
965
487
11,483
16,821
32,316
45,809
305-55
410
3,952
18,003
2,523
43,816
5,854
3,849
10,302
305-60
1,466
4,380
5,333
433
37,971
1,137
51,161
305-64
1,706
6,684
1,625
12,889
41,198
11,886
52,259
305-66P
305-68
16,439
14,863
305-69
1,448
12,670
382
49,477
2,548
50,678
305-70
610
3,041
12,903
8,123
20,481
Group II: AIDSVAX B/E
305-02
1,274
10,882
8,939
2,090
1,107
26,129
427
65,280
305-03
414
1,226
2,725
8,383
814
27,769
9,260
13,239
305-07P
305-11
19,669
5,467
26,877
3,578
305-12
15,579
623
3,764
33,730
4,385
22,335
305-20
439
511
1,235
3,132
667
9,218
8,314
305-21
2,316
9,786
15,794
55,467
1,892
65,298
305-22
17,708
16,269
1,451
57,446
14,396
2,396
53,839
305-24
312
747
14,691
25,485
305-35
526
10,319
5,687
11,698
240
61,381
22,146
46,337
305-36P
3,441
305-39
625
2,871
3,513
1,097
1,501
14,886
305-40
6,922
445
3,682
411
41,774
4,729
11,929
305-45P
305-46
6,942
47,847
31,940
305-47P
305-48
3,604
661
337
35,015
1,578
5,374
305-50P
305-51
2,641
1,415
10,841
51,506
2,914
34,386
305-52
213
5,254
2,328
7,727
51,910
2,398
622
53,033
305-56P
305-58
15,062
1,633
12,397
42,816
602
7,280
305-62
307
33,099
3,914
6,593
23,319
1,591
305-63
6,488
986
2,303
20,861
16,166
Binding Magnitude
(Maximum Binding)
Maximum binding: highest binding to a single peptide within an epitope identified.
Baseline binding subtraction: Binding intensity of corresponding RV144 baseline plasma to each peptide + 3stdev.
PSubjects who received placebo treatment.
PubID 
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Group III: ALVAC
305-06 1
1,101
1,656
1,477
305-09
305-15
1,043
966
3,462
305-17
373
305-18P
305-23
631
1,406
305-26P
926
1,810
574
316
1,321
942
423
796
305-29
305-31
4,229
305-32
205
1,357
305-33
334
430
305-37P
305-38
1,327
305-41
473
1,670
305-43
305-49
8,984
305-53P
305-54
617
305-57
454
524
3,701
305-59
567
6,740
305-61
224
3,427
305-65
269
536
504
1,208
305-67
1,473
1,089
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

9. Group I: ALVAC + AIDSVAX B/E
PubID
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Group I: ALVAC + AIDSVAX B/E
305-01
0.0
11.3
21.2
57.5
9.9
305-04
2.3
9.2
1.3
65.1
22.1
305-05
3.5
1.9
15.7
57.1
2.2
19.6
305-08
7.6
7.8
8.1
15.6
6.8
39.6
14.4
305-10
76.6
13.4
305-13
2.9
12.5
2.4
13.7
55.7
305-14
8.0
5.6
3.2
0.3
41.3
1.5
40.1
305-16P
11.1
305-19P
305-25
1.6
8.3
63.2
9.1
6.6
305-27
0.6
3.6
5.4
1.8
24.3
28.7
0.8
34.8
305-28
16.5
67.6
13.5
305-30
10.2
5.5
63.7
14.6
305-34
5.2
11.4
67.1
12.3
4.0
305-42
15.4
3.9
7.2
0.2
46.3
0.4
26.5
305-44
0.9
0.5
10.6
30.0
42.5
305-55
4.5
20.3
2.8
49.4
4.3
11.6
305-60
1.4
37.3
1.1
50.2
305-64
10.1
32.1
9.3
40.7
305-66P
305-68
52.5
47.5
305-69
1.2
10.8
42.2
43.2
305-70
6.7
28.6
18.0
45.3
Group II: AIDSVAX B/E
305-02
9.4
7.7
1.0
22.5
56.2
305-03
13.1
43.5
14.5
20.7
305-07P
305-11
35.4
9.8
48.3
6.4
305-12
19.4
4.7
41.9
27.8
305-20
13.3
39.2
305-21
6.5
10.5
36.8
43.4
305-22
35.1
8.8
32.9
305-24
35.6
61.8
305-35
7.4
38.8
14.0
29.3
305-36P
99.8
305-39
2.6
11.7
14.3
6.1
60.8
305-40
5.3
59.8
17.1
305-45P
305-46
55.2
305-47P
305-48
0.7
75.2
3.4
11.5
305-50P
305-51
2.5
49.7
33.2
305-52
6.3
42.0
42.9
305-56P
305-58
18.6
2.0
15.3
52.9
9.0
305-62
48.1
5.7
9.6
33.9
305-63
13.9
2.1
4.9
44.6
34.5
Percent of
Total Response
Percent of Total Response
maximum binding intensity for the epitope
sum of the max binding of all identified epitopes
= 100 x
PSubjects who received placebo treatment.
PubID
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Group III: ALVAC
305-06
0.0
26.0
39.1
34.8
305-09
11.1
305-15
19.0
17.6
63.2
305-17
0.3
97.9
305-18P
305-23
30.9
68.8
305-26P
13.0
25.5
8.1
4.4
18.6
13.2
6.0
11.2
305-29
305-31
99.8
305-32
0.1
13.1
86.5
305-33
43.3
55.8
305-37P
305-38
99.4
305-41
22.0
77.7
305-43
305-49
99.9
305-53P
305-54
0.2
98.7
305-57
9.7
79.0
305-59
7.8
92.2
305-61
6.1
93.7
305-65
10.7
21.3
20.0
47.9
305-67
57.3
42.4
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

10. Positivity rate = Number of IDs with positive binding to epitope
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Group 1
26.1
56.5
65.2
82.6
43.5
87.0
21.7
39.1
Group 2
29.2
62.5
58.3
75.0
25.0
33.3
79.2
Group 3
13.0
8.7
4.3
60.9
Number of IDs with positive binding to epitope
Total number of IDs in the group
Positivity rate =
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

11. Groups 1 and 2 Show Higher Magnitude of Env-Binding than Group 3
Sum of Max Binding = Sum of maximum binding intensities for all epitopes identified.
Statistical Analysis: Two-Tailed Mann Whitney U-test
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

12. Binding to C1/V1 and V2
Maximum Binding Intensity = Highest binding signal to a single peptide within the epitope region
Statistical Analysis: Two-Tailed Mann Whitney U-test
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

13. Binding to V3 and C5.2
Maximum Binding Intensity = Highest binding signal to a single peptide within the epitope region
Statistical Analysis: Two-Tailed Mann Whitney U-test
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

14. Relative Percentages of Specificity Across gp120 - Group Mean
C1.1
C1.2
C1/V1 V2 C2 V3 C4
C5.1
C5.2
Group 1
1.6
2.6
3.4
9.9
1.5
49.1
4.6
1.7
25.5
Group 2
2.1
8.3
6.9
1.1
38.6 4
34.8
Group 3 2
8.5
1.3
14.2
0.3
59.9
0.8
11.3
Percent of Total Response
maximum binding intensity for the epitope
sum of the max binding of all identified epitopes
= 100 x
Relative percentages across Env within an individual’s response are calculated; then the mean percentage values of all positive PTIDs in each group are plotted in these pie charts.
N = 17
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

15. Anti-gD Binding Magnitude is Greater in Groups 1 and 2
Group I: ALVAC + AIDSVAX B/E
Group II: AIDSVAX B/E
Group III: ALVAC
PubID
Max Binding
305-01
19,653
305-02
30,508
3006
12,367
305-04
55,776
305-03
40,772
3009
15,111
305-05
25,956
305-07P
1,851
3016 1
305-08
56,671
305-11
42,660
3018
6,470
305-10
7,539
305-12
52,838
3019
1,247
305-13
65,293
305-20
44,127
3024
39,354
305-14
52,135
305-21
61,714
3027
3,112
305-16P
9,676
305-22
64,063
3030
305-19P
305-24
53,476
3032
12,462
305-25
25,330
305-35
65,290
3033
305-27
30,168
305-36P
3034
2,433
305-28
46,308
305-39
21,230
3038
13,974
305-30
30,364
305-40
25,575
3039
5,467
305-34
43,276
305-45P
3042
11,419
305-42
65,098
305-46
63,890
3044
305-44
16,455
305-47P
3050
305-55
60,006
305-48
8,913
3054
305-60
56,263
305-50P
47,852
3055
8,367
305-64
55,386
305-51
64,859
3058
36,312
305-66P
305-52
62,660
3060
15,955
305-68
34,279
305-56P
3062
305-69
64,504
305-58
63,373
3066
50,987
305-70
29,256
305-62
38,360
3068
305-63
31,051
91.3%
87.5%
65.2%
positivity rate
Note: Group III did not receive AIDSVAX in RV305. Binding to gD peptides in this group should represent the binding response maintained from AIDSVAX boosts in RV144.
Max Binding = Highest signal intensity to a single peptide of the sequences covering the gD protein
Statistical Analysis: Two-tailed Mann-Whitney U-test
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC

16. Binding to 18 gD Peptides in Array Library Group Average
Sequence
gD 01
MGGAAARLGAVILFV
gD 02
AAARLGAVILFVVIV
gD 03
RLGAVILFVVIVGLH
gD 04
AVILFVVIVGLHGVR
gD 05
LFVVIVGLHGVRGKY
gD 06
VIVGLHGVRGKYALA
gD 07
GLHGVRGKYALADAS
gD 08
GVRGKYALADASLKM
gD 09
GKYALADASLKMADP
gD 10
ALADASLKMADPNRF
gD 11
DASLKMADPNRFRGK
gD 12
LKMADPNRFRGKDLP
gD 13
ADPNRFRGKDLPVLD
gD 14
NRFRGKDLPVLDQLL
gD 15
RGKDLPVLDQLLEVP
gD 16
DLPVLDQLLEVPVWK
gD 17
VLDQLLEVPVWKEAD
gD 18
QLLEVPVWKEADTTL
Plotted are the mean binding intensity values for each group to each of the 18 gD peptides that are included in the library.
Duffy/Shen, Tomaras Lab, DHVI, CAVIMC