2005-03-03220C Moore (revised)
SUPPLEMENTARY INFORMATION
Protection of macaques from vaginal SHIV challenge by
vaginally delivered inhibitors of virus-cell fusion
Ronald S. Veazey (1), Per Johan Klasse (2), Susan M. Schader (2), Qinxue Hu (3),
Thomas J. Ketas (2), Min Lu (4), Preston A. Marx (1), Jason Dufour (1), Richard J.
Colonno (5), Robin J. Shattock (3), Martin S. Springer (6), John P. Moore (2, #)
Supplementary Information
Synergy between entry inhibitors in vitro
To evaluate the suitability of CMPD167, BMS-378806 and C52L for use in
combination, we tested them in pairs against SHIV-162P3 infection of Tzm.bl cells and
HIV-1BaL infection of MDM1,2. Tzm-bl (formerly JC-53.bl) cells are derived from the
HeLa epithelial cell line. They express CD4 and CCR5 as transfected genes, and contain
inducible luciferase and ß-gal genes under the control of the HIV-1 LTR1. Inhibitors were
titrated alone and in combination at fixed ratios. The approximately monotonous zone of
10-99% inhibition was analyzed using the Calcusyn algorithm (Biosoft, Cambridge, UK).
Although the experimental variation was greater in the studies of SHIV-162P3
infection of Tzm.bl cells than of HIV-1BaL infection of MDM, both experimental systems
generated the same pattern of results. Thus, combining BMS-378806 with CMPD167
lead to approximately additive inhibition, whereas combinations of C52L with either
CMPD167 or BMS-378806 tended more towards synergistic inhibition (Supplementary
Table 1). These observations are consistent with other reports of synergy between
different classes of entry inhibitors, particularly those involving T-20, a peptide with the
same mechanism of action as C52L3,4.
Vaginal challenges with SHIV-162P4
We performed pilot studies using low concentrations of CMPD167 or C52L and
the SHIV-162P4 virus before switching to SHIV-162P3 to permit cross-comparison of
our results with those being generated by others5. The SHIV-162P4 stock was cultured at
the Tulane National Primate Research Center using seed virus provided by Cecelia
Cheng-Meyer (Rockefeller University); it infected 17/18 control animals (94%).
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Vaginally administered CMPD167 at 1mM was not protective against SHIV-162P4, as
only 2/16 animals remained uninfected in published and subsequent experiments6. In
contrast, C52L was partially protective when delivered vaginally in the range 0.050.5mM, with 4/10 animals remaining uninfected (p = 0.041, compared to control)
(Supplementary Fig.1).
Plasma VL in SHIV-162P3-infected animals
We compared plasma VL in the 9 control animals and in 13 that became infected
with SHIV-162P3 despite receiving inhibitors vaginally. The VL profiles in individual
animals varied (the error bars show the SD within the groups), but the mean values in the
control and inhibitor-treated animals were not significantly different at any time point
(Supplementary Fig.2). There were no significant differences between mean VL peak,
time to reach the peak, or Area Under the Curve (AUC) for the two groups as compared
by Student’s t test (Excel, Microsoft): peak height, 6.4 ± 0.44 log10 RNA copies/ml for
controls vs 6.3 ± 0.53 log10 RNA copies/ml for inhibitor-treated; peak time, 20 ± 5.5 days
vs 19 ± 4.2 days; AUC for days 7-42, 155 ± 13 vs 156 ± 24, p > 0.35. (The AUC of the
plot of log VL as a function of time was calculated by Prism (Graphpad)). This finding
suggests that any initial effect of the vaginally delivered inhibitors on reducing the
number of virus particles that are successfully transmitted across the vaginal mucosa is
insignificant after viral amplification takes place within lymphoid tissues. An analogy
might be to the lack of relationship between inoculum size and the outcome of infection
when macaques are infected with SIV strains that, like SHIV-162P3, use CCR5 for
entry7.
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Supplementary references
1. Wei, X. et al. Emergence of resistant human immunodeficiency virus type 1 in patients
receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 46, 18961905 (2002).
2. Hu, Q. et al. Blockade of attachment and fusion receptors inhibits HIV-1 infection of
human cervical tissue. J. Exp. Med. 199, 1065-1075 (2004).
3. Tremblay, C. Effects of HIV-1 entry inhibitors in combination. Curr. Pharm. Des. 10,
1861-1865 (2004).
4. Reeves, J.D. et al. Sensitivity of HIV-1 to entry inhibitors correlates with
envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc. Natl. Acad. Sci.
USA 99, 16249-16254 (2002).
5. Lederman, M.M. et al. Prevention of vaginal SHIV transmission in rhesus macaques
through inhibition of CCR5. Science 306, 485-487 (2004).
6. Veazey, R.S. et al. Use of a small-molecule CCR5 inhibitor in macaques to treat
simian immunodeficiency virus infection and prevent simian-human immunodeficiency
virus infection. J. Exp. Med. 198, 1551-1562 (2003).
7. Igarashi, T. et al. Early control of highly pathogenic simian immunodeficiency
virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys
usually results in long-lasting asymptomatic clinical outcomes. J. Virol. 77, 10829-10840
(2003).
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Legends for Supplementary Figures
Supplementary Fig.1
Challenge studies with SHIV-162P4.
Each symbol represents a single macaque,
grouped by infection status post-challenge with SHIV-162P4. Control animals (triangles),
inhibitor-treated animals (diamonds). All animals received HMC gel (pH 7.0), containing
or lacking inhibitors, except for those denoted by yellow bordered-symbols (PBS use).
Eleven of the animals receiving CMPD167 (1mM) have been previously described; of
them, 2 were uninfected6.
Supplementary Fig.2
Plasma VL in SHIV-162P3-infected animals. The mean VL values (log10 RNA copies
per ml of plasma, ± standard deviation) are plotted for the first 42 days post challenge.
The 9 infected control animals (red circles) are compared with 13 infected animals that
had received vaginally delivered inhibitors (blue squares). A dashed green line at
log10(125) RNA copies per ml indicates the limit of sensitivity of the VL assay.
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Supplementary Table 1. Combination indices (CI)1 at 50, 75 and 90% effective doses
(ED) for inhibitor combinations in vitro
Virus and cells Compounds
ED50
ED75
ED90
(molar ratio)
HIV-1 BaL
CMPD167 +
0.96 ± 0.055 1.0 ± 0.065 1.1 ± 0.065
Macrophages
BMS-378806
(1/10)
CMPD167 +
0.47 ± 0.010 0.50 ± 0.045 0.58 ± 0.075
C52L
(1/5)
BMS-378806 +
0.56 ± 0.050 0.64 ± 0.050 0.73 ± 0.050
C52L
(2/1)
SHIV-162P3
CMPD167 +
1.4 ± 0.38
1.2 ± 0.25
1.1 ± 0.23
Tzm-b1 cells
BMS-378806
(1/90)
CMPD167 +
0.74 ± 0.15 0.57 ± 0.094 0.47 ± 0.079
C52L
(1/3.2)
BMS-378806 +
0.83 ± 0.23 0.56 ± 0.050 0.41 ± 0.10
C52L
(3.3/1)
1
CI < 1 indicates synergy, = 1 additivity, > 1 antagonism. The formula used applies to
mutually exclusive inhibitors: CIEDx=[((D1)comb/(D1))+ ((D2)comb/(D2))], where (D1) and
(D2) are the concentrations of the respective drug on its own needed to achieve X %
inhibition; (D1)comb and (D2)comb are the respective concentrations of the two drugs, when
used in combination, needed to give the same level of inhibition. The CI values given are
the means ± SD of 2-3 experiments. The r-values for describing the degree of fit to the
dose-effect equation in Calcusyn were > 0.95.
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