Supporting Information
Fouts et al. 10.1073/pnas.1423669112
b.
a.
Naïve
rhFLSC
Gp120
rhFLSC
RC529
Gp120
Iscomatrix
c.
d.
IFN-γ SFC </= Mean (n=14)
Challenges
to Infection
5
11-12
IFN-γ SFC >/= Mean (n=10)
0.8
7-8
Log ADCC
Fraction Uninfected
1.0
0.6
0.4
p = 0.325
6-7
4-5
4
3-4
1-2
0.2
3
0.0
0
2
4
6
8
10
12
14
Number of Intrarectal Challenges
2
1000
800
600
400
200
0
IL-2 ELISPOT
e.
Immunogen
Adjuvant
Animal #
Naïve
Naïve
Naïve
Naïve
Naïve
Naïve
gp120
gp120
gp120
gp120
gp120
gp120
rhFLSC
rhFLSC
rhFLSC
rhFLSC
rhFLSC
rhFLSC
gp120
gp120
gp120
gp120
gp120
gp120
rhFLSC
rhFLSC
rhFLSC
rhFLSC
rhFLSC
rhFLSC
None
None
None
None
None
None
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
RC529-SE
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
Iscomatrix
A1N099
A3N049
A3N058
A3N100
A3N107
A4N053
A2N041
A3N057
A3N061
A3N104
A4N037
A4N078
A3N052
A3N065
A3N078
A4N030
A4N051
A4N079
A3N064
A3N068
A3N072
A3N085
A4N036
A4N046
A3N071
A3N080
A3N086
A3N109
A4N042
A4N049
Clade B Tier 1 isolates
MN
SF162.LS
BaL.26
<20
<20
<20
<20
<20
<20
500
155
200
600
1188
866
7010
183
1992
717
947
239
370
5599
2641
2432
3499
843
43740
4751
5198
5198
1956
1225
<20
<20
<20
<20
<20
<20
226
39
115
191
366
108
5668
73
597
405
188
129
129
1055
1100
87
813
372
9566
712
1364
1364
410
567
<20
<20
<20
<20
<20
<20
41
21
30
42
56
62
176
<20
93
44
<20
34
27
226
116
33
89
175
348
130
134
134
48
89
>20-100
>100-1000
>1000-10,000
>10,000
Fig. S1. (A) Longitudinal IFN-γ and IL-2 ELISPOT responses (spot-forming cells; SFC) to HIV-1Ba-L gp120 peptides. The asterisk indicates a significantly higher
ELISPOT response with Iscomatrix versus RC529 adjuvants for the same antigen (P < 0.05, t test). Week 63 is the day of challenge. (B) Polyfunctional FACS
analysis of PBMCs harvested at week 26, 2 wk after the third inoculation. Percentages inside the pie represent the total number of T cells responding to the
HIV-1Ba-L gp120 peptide stimulation. Polyfunctionality was assessed against IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a as described (1, 2). The colored portions of
the pie represent the portion of the T-cell response presenting a single function (red), two functions (green), or three functions (blue). Data shown are those
where the total T-cell response was greater than background measured with vaccine-naive control macaques. (C) Comparison of the rate of acquisition between animals dichotomized by the mean IFN-γ ELISPOT responses (SFC) measured at week 61 (2 wk after the final boost). Data were analyzed by log-rank test.
(D) Comparisons of IL-2 versus log ADCC responses (measured at the time of first challenge) versus acquisition for all study 1 animals. The two measures are
plotted for each animal (circles). The circles are color-coded according to the number of challenges needed to infect the animal. (E) Neutralizing of tier 1
pseudoviruses by sera collected at week 26 (2 wk after the third inoculation) in a TZM-bl–based assay (main text). Values presented are the maximum serum
dilution at which relative luminescence units were reduced by 50% compared with virus control wells without serum. The titer values are shown and colorcoded (right box). An MuLV pseudotyped virus served as control for nonspecific effects and was not neutralized (not shown).
1. Betts MR, et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281(1-2):65–78.
2. Xu R, et al. (2008) Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines. Vaccine 26(37):4819–4829.
Fouts et al. www.pnas.org/cgi/content/short/1423669112
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a.
Half-maximal Competition
Binding Titer
b.
Fig. S2. Dose escalation study. (A) Comparison of half-maximal serum-binding titers to rhFLSC or HIV-1Ba-L gp120 in studies 1 and 2, compared with an
unpublished dose escalation study (with 300, 600, or 1,200 μg rhFLSC) and a previously reported study that used QS21 adjuvant (1). Mean group titers are
shown; bars indicate SD. In all studies, titers were measured by antigen capture ELISA 2 wk after the final immunization as previously described (1). The amount
of antigen and/or adjuvant used is shown. (B) Comparison of cumulative competition binding titers against CD4i epitopes (17b, 19e, A32) measured as previously described (1). Mean group titers are shown; bars indicate SD.The immunization groups are the same as in A. Pairwise group comparisons were made by
Mann–Whitney–Wilcoxon test. In the dose escalation study, the group that received 1,200 μg of rhFLSC exhibited the highest cumulative titer compared with
the other two groups (P = 0.013).
1. DeVico A, et al. (2007) Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens. Proc Natl Acad Sci
USA 104(44):17477–17482.
Fouts et al. www.pnas.org/cgi/content/short/1423669112
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Fig. S3. Tat and Tat toxoid binds to rhFLSC. Tat toxoid was mixed with rhFLSC at the same concentrations used to formulate the vaccine (Fig. 1) and then crosslinked with BS3 as previously described (1). RhFLSC and HIV-1 Tat alone were treated with cross-linker as controls. Cross-linked preparations were analyzed by
Coomassie-stained SDS/PAGE (A) and anti-rhFLSC (B) or anti-Tat (C) Western blots. RhFLSC, Tat, and Tat toxoid were run as controls for comparison under
reducing and nonreducing conditions. Recognition of the same higher molecular weight bands (arrows) in cross-linked material by both the anti-rhFLSC and
anti-Tat antibodies in lanes 3 and 4 indicates that Tat and Tat toxoid associates with rhFLSC.
1. DeVico A, Silver A, Thronton AM, Sarngadharan MG, Pal R (1996) Covalently crosslinked complexes of human immunodeficiency virus type 1 (HIV-1) gp120 and CD4 receptor elicit
a neutralizing immune response that includes antibodies selective for primary virus isolates. Virology 218(1):258–263.
Fouts et al. www.pnas.org/cgi/content/short/1423669112
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a.
Total SIVmac239 Env-Specific Responses
Total SIVmac239 Gag-Specific Responses
b.
Fig. S4. (A) Polyfunctional FACS analysis of animals in study 3 using samples collected at week 12, 2 wk after the final DNA immunization. Responses were
measured with SIVmac239 Env or Gag peptides. Polyfunctionality was assessed against IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a (1, 2). The colored portions of the
pie represent the portion of the T-cell response presenting a single function (red), two functions (green), three functions (blue), or four functions (orange). The
number outside the pie represents the percentage of the total response that has two or more functions. Data shown are those where the total T-cell response
was greater than background measured with vaccine-naive control macaques. (B) Neutralization titers (EC50) measured in the TZM-bl assay using pseudoviruses
with tier 1A SIVmac251.6, tier 1B SIVsmE660/BR-CG7V, and tier 2 SIVmac251.3. The data shown were generated from samples taken 2 wk after the subunit or
sham boost (Fig. 1). Values presented are the maximum serum dilutions at which infection was reduced by 50% (ID50) compared with virus control wells
without serum. Mean group values are shown; bars indicate SD. An MuLV pseudotyped virus served as control for nonspecific effects and was not neutralized
(not shown).
1. Betts MR, et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281(1-2):65–78.
2. Xu R, et al. (2008) Comparative ability of various plasmid-based cytokines and chemokines to adjuvant the activity of HIV plasmid DNA vaccines. Vaccine 26(37):4819–4829.
Fouts et al. www.pnas.org/cgi/content/short/1423669112
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