Antibody Conjugation Approach Enhances
Breadth and Potency of Neutralization of
Anti-HIV-1 Antibodies and CD4-IgG
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Julia Gavrilyuk, Hitoshi Ban, Hisatoshi Uehara, Shannon J.
Sirk, Karen Saye-Francisco, Angelica Cuevas, Elise
Zablowsky, Avinash Oza, Michael S. Seaman, Dennis R.
Burton and Carlos F. Barbas III
J. Virol. 2013, 87(9):4985. DOI: 10.1128/JVI.03146-12.
Published Ahead of Print 20 February 2013.
Antibody Conjugation Approach Enhances Breadth and Potency of
Neutralization of Anti-HIV-1 Antibodies and CD4-IgG
Departments of Chemistry and Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USAa; Dainippon
Sumitomo Pharma, Osaka, Japanb; Mitsubishi Chemical Group Science and Technology Research Center, Yokohama, Japanc; Department of Immunology and Microbial
Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California, USAd; Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts, USAe
Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the
neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of
virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective
functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly
alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of
a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize
100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1
strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1
therapies.
I
nnovative new approaches to HIV-1 prophylaxis and therapy
are desperately needed. Despite the successes of highly active
antiretroviral therapy (HAART), more than 2 million people die
each year and more than 33 million individuals are infected
worldwide (http://aids.gov/hiv-aids-basics/hiv-aids-101/global
-statistics/). Although HAART is typically effective, it is not without problems, including complicated drug-drug interactions, adherence issues, and a myriad of side effects. The development of
potent and broadly acting biologic drugs might offer a solution to
some of these problems and complement traditional HAART.
Broadly neutralizing monoclonal antibodies (BNmAbs) that
recognize features conserved across clades of HIV are promising
starting points for the development of immunotherapeutic agents
against HIV-1 (1–8). Several studies have shown that the transfer
of sufficient quantities of broadly neutralizing antibodies can
achieve sterilizing immunity against intravenous, vaginal, or rectal challenge in macaque models (9, 10). The delivery of broadly
neutralizing antibodies using gene-based approaches has also
been shown to be effective in animal models (11, 12). Indeed, soon
after our discovery of BNmAb b12, we developed protein engineering methods to increase the potency and breadth of neutralization by b12 with the original aim of developing evolved b12
variants for HIV-1 therapy (13, 14). Collectively, these studies
suggest that BNmAbs could be effective HIV-1 prophylactic and
therapeutic agents. Unfortunately, even the most broadly neutralizing antibody is vulnerable to viral escape, because a single amino
acid change on the target protein can alter the binding epitope. If
a BNmAb could be modified to inhibit HIV in multiple ways, the
evolutionary hurdle for escape would be significantly elevated.
Furthermore, by combining multiple inhibitory functions in a
single molecule, the regulatory and cost issues for a biologic complement to combinatorial drug therapy might be minimized.
Recently, we developed a new class of therapeutic molecules by
May 2013 Volume 87 Number 9
demonstrating that catalytic monoclonal antibodies covalently
linked to designed ligands possess potent biological activities in a
variety of animal models of disease (15–19). Several of these are
now in clinical development (20). These studies revealed the many
advantages of coupling active small molecules and peptides with
antibodies. In contrast to bispecific-antibody approaches based
on protein engineering, such as the dual-variable-domain
(DVD)-Ig (21) or single-chain variable fragment (scFv)-Ig (22)
fusion approaches, among others, laborious protein engineering
is not required to endow a second specificity when the desired
ligand is chemically coupled to the antibody. Furthermore, expression issues are bypassed, since development of a new cell line
is not required.
A promising additional blockade to HIV-1 infection that
should complement the targeting of viral proteins is the targeting
of host proteins required for viral entry and replication. Unlike
viral proteins, host proteins are not under selective pressure to
evolve to evade the therapeutic agent. A number of small-molecule inhibitors of the HIV-1 coreceptors CCR5 and CXCR4 have
been developed (23, 24), and one CCR5-targeting drug has been
approved (25–28). Here we covalently linked a CCR5-targeting
small molecule, aplaviroc, to BNmAbs and CD4-IgG. This approach provided rapid access to bispecific proteins with excep-
Received 9 November 2012 Accepted 2 February 2013
Published ahead of print 20 February 2013
Address correspondence to Carlos F. Barbas III, carlos@scripps.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.03146-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.03146-12
Journal of Virology
p. 4985– 4993
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4985
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Julia Gavrilyuk,a Hitoshi Ban,a,b Hisatoshi Uehara,a,c Shannon J. Sirk,a Karen Saye-Francisco,d Angelica Cuevas,d Elise Zablowsky,e
Avinash Oza,e Michael S. Seaman,e Dennis R. Burton,d Carlos F. Barbas IIIa
Gavrilyuk et al.
tional breadth in their abilities to neutralize diverse isolates of
HIV-1.
MATERIALS AND METHODS
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Antibodies. Antibodies b12, 2G12, and DEN3 were provided by Dennis
R. Burton (Scripps Research Institute); antibodies PG9 and PG16 were
provided by the IAVI; the CD4-IgG2 immunoadhesion protein was obtained from Progenics (PRO542). Antibodies were stored at 4°C. Therapeutic-grade trastuzumab (Genentech) was used without additional purification.
Synthesis of labeling reagents. The synthesis and characterization of
aplaviroc with a linker have been described previously (29).
Antibody labeling procedure. In a 1.5-ml tube, an antibody solution
(99 ␮l; 1.5 mg/ml in 0.1 M Na2HPO4 [pH 8.0]) and 10 equivalents of
4-formylbenzene diazonium hexafluorophosphate (FBDP; 1 ␮l; 10 mM
solution in CH3CN) were combined (30). The solution was mixed gently
and was allowed to react for 30 min at room temperature with intermittent mixing. The solution turned yellow upon completion of the reaction.
After 30 min, excess FBDP was removed using a Zeba Spin desalting
column (molecular weight cutoff [MWCO], 7,000 [7K]; Pierce), and buffer was exchanged with 0.1 M Na2HPO4 (pH 6.0). Aplaviroc-oxyamine
(20 equivalents; 2 ␮l; 10 mM solution in CH3CN) was added, and the
solution was incubated overnight at 4°C. Excess aplaviroc-oxyamine was
removed using a Zeba Spin desalting column (MWCO, 7K), and free
aplaviroc was removed by using a protein A spin column (GE Healthcare)
according to the manufacturer’s instructions.
Tryptic digestion and quadrupole time of flight tandem mass spectrometry (Q-TOF MS-MS) characterization of antibody conjugates.
Purified antibody samples (100 ␮l; 1 mg/ml) were exchanged into 6 M
guanidine, 0.1 M Tris (pH 8.0) by using 0.5-ml Zeba Spin desalting columns (MWCO, 7K) according to the manufacturer’s instructions. To
each sample, 1 M dithiothreitol (DTT) (Fisher) was added to a final concentration of 20 mM. The samples were incubated for 1 h at 37°C with
gentle shaking, and a 1 M solution of iodoacetamide (Fisher) was added to
each sample to a final concentration of 40 mM. Samples were incubated at
room temperature in the dark for 40 min. The alkylation reactions were
quenched by adding 1 M DTT to a 40 mM final concentration. Sample
buffer was then exchanged for trypsin digestion buffer (50 mM Tris [pH
7.5], 5 mM CaCl2) using 0.5-ml Zeba Spin desalting columns (MWCO,
7K). Trypsin Gold (0.5 mg/ml; Pierce) was dissolved in 50 mM acetic acid.
The trypsin solution was added to each sample at an enzyme-to-protein
ratio of 1:20 (wt/wt), and samples were incubated at 37°C with shaking at
600 rpm for approximately 18 h. Digestion was stopped by the addition of
trifluoroacetic acid (TFA) to approximately 0.1%.
Samples (with 20 ␮g protein injected) were analyzed using an Agilent
6510 Q-TOF mass spectrometer equipped with a Zorbax SB C18 column
(narrow bore; inner diameter, 2.1 mm; length, 150 mm; particle size, 3.5
␮m) (Agilent). High-performance liquid chromatography (HPLC) parameters were as follows: flow rate, 0.2 ml/min; a gradient from 0 to 40%
mobile phase B over 80 min, followed by a gradient to 0% B from 80 to 90
min. Mobile phase A was 0.05% TFA (vol/vol) in HPLC-grade H2O, 2%
acetonitrile (vol/vol), and mobile phase B was 0.04% TFA (vol/vol) in
90% acetonitrile (vol/vol). MS data were collected for 200 to 2,500 m/z
and 100 to 2,000 m/z, positive polarity, a gas temperature of 325°C, a
nebulizer pressure of 30 lb/in2, and a capillary voltage of 3,500 V. Data
were analyzed using MassHunter software (Agilent) and GPMAW software (version 8.20; ChemSW).
Flow cytometry. Flow cytometry experiments were performed as described previously (29). The cell lines used were A431 cells, which do not
express CCR5, and TZM-bl cells, which do express CCR5. In brief, a
single-cell suspension was prepared, and cells were washed twice with cold
stain buffer (BD Pharmingen) and were pelleted by centrifugation (300 ⫻
g). The cell pellet was resuspended in cold stain buffer to a final concentration of 2 ⫻ 107 cells/ml. Aliquots of 50 ␮l were distributed to V-bottom
wells of microwell plates (Corning). Primary antibodies were added to a
final concentration of 20 ␮g/ml; each sample was tested in triplicate. The
cells were incubated on ice for 1 h, washed twice with stain buffer, and
resuspended in 100 ␮l of stain buffer. A fluorescence-labeled secondary
antibody was added at a concentration recommended by manufacturer.
The plate was incubated on ice, protected from light, for 1 h. The cells were
washed twice with the stain buffer, resuspended in 200 ␮l of stain buffer,
and transferred to filter-top fluorescence-activated cell sorter (FACS)
tubes (BD Biosciences) containing 300 ␮l of buffer (2% fetal bovine serum [FBS] in phosphate-buffered saline [PBS]). Cell counting was performed using a digital LSR II cytometer. Data were analyzed with FlowJo
software, version 8.7.1.
Pharmacokinetic study. The pharmacokinetic experiment was carried out as described previously (19). In brief, all animal experiments were
performed according to Division of Animal Resources (TSRI) guidelines
following approved protocols. Female athymic nude mice (8 weeks of age)
were injected subcutaneously with 100 ␮g trastuzumab or trastuzumabaplaviroc in 100 ␮l sterile PBS (4 animals per group). Blood was collected
from tail veins after 5 min, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 168 h, and
336 h. In order to minimize blood loss, only 10 ␮l of blood was withdrawn
per bleed; blood was diluted 1/5 in PBS containing 1% bovine serum
albumin (BSA). Samples were allowed to chill on ice for 20 min. Insoluble
components were removed by centrifugation, and samples were stored at
⫺20°C until analysis.
The binding and wash buffer for enzyme-linked immunosorbent assays (ELISA) was PBS containing 1% BSA. Half-area ELISA plates (Corning) were coated with anti-human IgG Fc (Pierce) at 750 ng/well overnight at 4°C. After wells were blocked with 3% BSA, serum samples
diluted in binding buffer were added, and samples were incubated for 1 h
at 37°C. Trastuzumab and trastuzumab-aplaviroc were detected by incubation with donkey anti-human IgG conjugated with horseradish peroxidase (HRPO) (Jackson ImmunoResearch). To determine the serum dilution at which 80% saturation was reached, the 5-min samples were
serially diluted and analyzed; 80% saturation of the ELISA signal at time
zero was reached at a dilution of 1/500 for the anti-human IgG. In all
subsequent experiments, the optical density (OD) at time zero was set as
100%, and the absorption at all later time points was displayed as a percentage of that at time zero. The resulting curves were fitted using GraphPad Prism one-phase exponential decay.
Virus neutralization assay. Neutralization assays with single-round
infectious pseudovirus were performed as described elsewhere (10) by
using U87.CD4.CCR5 target cells obtained from the NIH AIDS Research
and Reference Reagent Program (contributed by HongKui Deng and Dan
Littman). Briefly, 1 ⫻ 104 target cells in a volume of 100 ␮l were seeded
into wells of 96-well plates (Corning) and were incubated overnight at
37°C under 5% CO2. After overnight incubation, the medium was removed, and 50 ␮l of fresh medium was added to each well. Serially diluted
samples (50 ␮l) were transferred to plated target cells and were incubated
for 1 h at 37°C. An equal volume of virus previously determined to yield
2 ⫻ 105 relative light units (RLU) was added (100 ␮l), and plates were
incubated for an additional 72 h. To measure luciferase activity, the medium was removed, the wells were washed once with Ca2⫹- and Mg2⫹free phosphate-buffered saline, and 50 ␮l of an appropriately diluted luciferase cell culture lysis reagent (Promega) was added and mixed by
pipetting vigorously up and down. Aliquots (20 ␮l) were transferred to
opaque 96-well assay plates (Corning), and luciferase activity was measured on a luminometer (EG&G Berthold LB96V; Perkin-Elmer) using a
luciferase assay substrate (Promega). The percentage of virus neutralization at a given antibody concentration was determined by calculating the
reduction in luciferase activity in the presence of an antibody (relative to
that in virus-only wells).
High-throughput virus neutralization assay. High-throughput
screening of the PG9-aplaviroc conjugate was performed in the laboratory
of Michael Seaman, Harvard Medical School. The highest concentration
of PG9-aplaviroc tested was 50 ␮g/ml; seven 5-fold dilutions were also
Potent, Broadly Active Bispecific Anti-HIV Antibodies
modified with the FBDP reagent at a surface-exposed tyrosine residue(s), introducing an aldehyde tag onto the antibody. In the second step, chemoselective
conjugation of the aldehyde with oxyamine is performed, resulting in the introduction of the aplaviroc moiety onto the surface of the antibody.
tested in duplicate wells. The highest concentration of aplaviroc tested was
100 nM.
RESULTS
Model trastuzumab-aplaviroc conjugate. In order to independently assess the activity of aplaviroc conjugated to a carrier IgG
without anti-HIV-1 activity, trastuzumab, an anti-Her2 monoclonal antibody (MAb), was used as a model human IgG for chemical modification and testing. Bioconjugation conditions were selected on the basis of our previous study (30) and were further
optimized here (for full optimization details, see the tables in section S3 in the supplemental material). The two-step conjugation
methodology involved modification of the IgG with FBDP (4formylbenzene diazonium hexafluorophosphate) followed by
ligation of the antibody to aplaviroc-oxyamine (Fig. 1). The trastuzumab-aplaviroc conjugate was subjected to extensive purification, including gel filtration and protein A purification, to ensure
complete removal of unreacted aplaviroc.
Characterization of trastuzumab-aplaviroc by matrix-assisted
laser desorption ionization–time of flight (MALDI-TOF) mass
spectrometry revealed the incorporation of an average of 1 aplaviroc moiety per IgG molecule (see the supplemental material).
Tryptic digestion and MS-MS characterization of the trastuzumab-aplaviroc indicated that the diazonium modification occurred preferentially at the conserved surface-exposed tyrosine in
the Fc domain at position 319 by Kabat numbering. Some minor
modification was also observed in the heavy chain of Fab (see the
supplemental material). This observation is in agreement with
previous studies that demonstrated preferential diazene formation with the most accessible tyrosines (22, 31, 32). When trastuzumab that was not modified with FBDP was incubated with aplaviroc-oxyamine, no conjugation of aplaviroc-oxyamine occurred,
as shown by MALDI-TOF mass spectrometry. This sample had no
activity in the HIV neutralization assays, indicating that the purification protocol used removed unconjugated aplaviroc-oxyamine.
Flow cytometry demonstrated that trastuzumab-aplaviroc
bound to Her2-positive A431 cells and, to a lesser extent, to
CCR5-positive TZM-bl cells (Fig. 2A). The parent antibody, trastuzumab, also bound to TZM-bl cells, due to low-level expression
May 2013 Volume 87 Number 9
of Her2; however, mean fluorescence intensity was considerably
higher for trastuzumab-aplaviroc bound to TZM-bl cells than for
the parent antibody, indicating that the CCR5 binding activity of
aplaviroc was maintained following conjugation.
The anti-HIV-1 activity of trastuzumab-aplaviroc was assessed
by neutralization assays with a single round of infectious pseudovirus. Trastuzumab-aplaviroc neutralized HIV-1 strains JR-FL
and YU2 with 50% inhibitory concentrations (IC50s) of 2.3 nM
and 4.9 nM, respectively; unmodified trastuzumab showed no
neutralizing activity (Fig. 2B). The potent CCR5 binding antibody
2D7 neutralized HIV-1 strains JR-FL and YU2 with IC50s of 0.2
nM and 0.1 nM, respectively. To ensure the CCR5-based mechanism of trastuzumab-aplaviroc, it was also tested for neutralization of CXCR4-tropic HIV HXB2 and was found to be inactive
(see the supplemental material).
Modification of the antibody occurred primarily at Tyr 319 in
the CH2 region of the heavy chain. Because the in vivo half-lives
(t1/2) of antibodies are mediated primarily by contacts in the CH2
region of the IgG with the neonatal Fc receptor FcRn, we evaluated
the half-lives of the parent and the conjugate in mice. The trastuzumab-aplaviroc conjugate had pharmacokinetic properties similar to those of the unmodified parent antibody (Fig. 2C). The
half-life of trastuzumab in athymic nude mice was 115 h, whereas
the half-life of the trastuzumab-aplaviroc conjugate was 168 h.
The half-life of aplaviroc in mice is 30 min (33). Therefore, conjugation of aplaviroc with trastuzumab dramatically extended the
half-life of aplaviroc in vivo and did not negatively impact the
half-life of the scaffold antibody.
Conjugation of aplaviroc to broadly neutralizing monoclonal antibodies and CD4-IgG. BNmAbs b12, 2G12, PG9, and PG16
and the immunoadhesin protein CD4-IgG were conjugated with
aplaviroc, and the conjugates were characterized by MALDI-TOF
mass spectrometry. We observed the incorporation of 0.5 to 2 aplaviroc moieties per protein molecule (Table 1). Tryptic digestion and
MS-MS analysis of conjugates indicated that the heavy-chain constant region Tyr 319 was the primary site of chemical modification in
each BNmAb, although minor modification sites were also identified
(see the supplemental material). No modifications were observed in
the Fv regions of the antibodies.
jvi.asm.org 4987
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FIG 1 Schematic representation of the chemical modification of an antibody with a diazonium hexafluorophosphate (FBDP) reagent. The antibody is first
Gavrilyuk et al.
HIV-1 neutralization studies showed that the potencies of
BNmAb conjugates against HIV-1 isolates JR-FL and YU2 were
significantly higher than those of the unconjugated parental antibodies or the CD4 fusion protein (Table 1; Fig. 3). Improvements
in potency over the parent antibody ranged from ⬃3-fold for
2G12-aplaviroc against the JR-FL isolate to ⬎400-fold for b12aplaviroc against the YU2 isolate. Differences between conjugates
and parent antibodies were smallest for the most potent of the
parental antibodies. In the neutralization assay with the JR-FL
TABLE 1 Neutralization of HIV-1 JR-FL and YU2 by aplavirocconjugated BNmAbs and CD4-Ig
a
Antibody
b12-apl
b12
2G12-apl
2G12
PG9-apl
PG9
PG16-apl
PG16
CD4-IgG–apl
CD4-IgG
Aplaviroc
2D7 (positive control)
DEN3 (negative control)
a
apl, aplaviroc.
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Avg no. of
aplaviroc moieties
per IgG molecule
0.5
0
2
0
1
0
2
0
1
0
0
0
IC50 (nM) for:
JR-FL
YU2
0.1
0.3
0.3
0.8
11.6
⬎100
5.9
⬎100
0.6
0.6
0.95
0.2
⬎1,000
0.1
46.7
25.5
⬎100
1.2
22.6
0.4
1.5
0.6
0.7
0.86
0.1
⬎1,000
isolate, PG9 and PG16 were inactive except as aplaviroc conjugates. CD4-IgG potently neutralizes both JR-FL and YU2. Conjugation of aplaviroc to CD4-IgG did not have a notable effect on the
neutralization of these viruses, since the immunoadhesin protein
itself can neutralize JR-FL and YU2 more potently than aplaviroc
alone. To determine whether aplaviroc conjugation can have a
positive effect on the activity of this protein, we additionally assayed the clade A variant 92RW 020.5, a strain relatively resistant
to neutralization by CD4-IgG. Against this strain, the conjugation
resulted in a 20-fold improvement in the IC50. CD4-IgG neutralized the 92RW pseudovirus with an IC50 of 10 nM, whereas CD4IgG–aplaviroc neutralized this isolate with an IC50 of 0.5 nM. Significantly, no evidence of enhanced infection was noted with any
conjugated protein studied.
We also studied the neutralization activity of a 1:1 molar
mixture of aplaviroc and PG9 against HIV-1 strains JR-FL and
YU2, and we observed a potency equal to that of aplaviroc. The
in vitro assay, however, does not allow us to assess the potential
clinical benefit of the extended half-life of the antibody-aplaviroc conjugate relative to the 1:1 mixture of two agents (vide
infra).
Neutralization of HIV-1 strains by PG9-aplaviroc. The PG9
conjugate was chosen for more in-depth study because the parent
antibody, PG9, is representative of a recently described group of
BNmAbs that demonstrate tremendous breadth of neutralization
activity. ELISA studies of PG9 and PG9-aplaviroc performed using gp120 from HIV strain 16055, a clade C virus, indicated no loss
of binding activity for PG9 following conjugation. The neutralizing ability of PG9-aplaviroc was assessed against a panel of 117
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FIG 2 Evaluation of the model antibody conjugate trastuzumab-aplaviroc. (A) Flow cytometry analysis of binding to CCR5-positive TZM-bl cells and
CCR5-negative A431 cells. The potent CCR5 binding antibody (Ab) 2D7 served as a positive control in TZM-bl cell assays. MFI, mean fluorescence intensity. (B)
Neutralization of HIV-1 JR-FL and YU2 by trastuzumab and the trastuzumab-aplaviroc conjugate. (C) Pharmacokinetic profiles of trastuzumab and the
trastuzumab-aplaviroc conjugate in athymic mice (4 mice per experimental group). All experimental data represent two or more independent experiments
performed in triplicate. Error bars represent standard deviations.
Potent, Broadly Active Bispecific Anti-HIV Antibodies
pseudoviruses representing major circulating HIV-1 subtypes
(Table 2; see also section S5 in the supplemental material). PG9
neutralized 101 of the 117 viruses tested with IC50s below 50 ␮g/
ml. The 16 viruses that were not well neutralized by PG9 were
neutralized by PG9-aplaviroc with a mean IC50 of 6 ␮g/ml (Table
3). PG9-aplaviroc neutralized all 117 of the subtypes with IC50s
below 17 ␮g/ml and with a mean IC50 28 ␮g/ml (see the supplemental material). PG9-aplaviroc neutralized 94.9% of the 117 vi-
TABLE 2 Summary of neutralization data for a panel of 117 HIV-1 strains
a
Aplaviroc neutralization with IC50s of ⬍100 nM and IC80s of ⬍100 nM.
May 2013 Volume 87 Number 9
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FIG 3 Evaluation of the neutralization abilities of antibody conjugates against HIV-1 strains JR-FL and YU2 and of the CD4-IgG2–aplaviroc conjugate against
HIV-1 strains JR-FL, YU2, and 92RW. HIV-1 JR-FL is resistant to neutralization by parent antibodies PG9 and PG16. The bottom right graph shows results for
controls only: 2D7, positive-control MAb; DEN3, negative-control MAb; aplaviroc, small-molecule positive control.
Gavrilyuk et al.
TABLE 3 Summary of the pseudoviruses neutralized by PG9-aplaviroc
with IC50s of ⬍50 ␮g/ml that are not neutralized by PG9 (IC50s, ⬎50
␮g/ml)
TABLE 4 Summary of the pseudoviruses neutralized by PG9-aplaviroc
with IC80s of ⬍50 ␮g/ml that are not neutralized by PG9 (IC80s, ⬎50
␮g/ml)
IC50 titer in TZM-bl cells
(␮g/ml)c
IC80 titer in TZM-bl cells
(␮g/ml)c
Cladeb
PG9
PG9-aplaviroc
Virus IDa
Cladeb
PG9
PG9-aplaviroc
QH0692.42
1054_07_TC4_1499
6244_13_B5_4576
62357_14_D3_4589
ZM214M.PL15
Ce1086_B2
Ce2010_F5
246F C1G
7030102001E5(Rev-)
CNE30
T251-18
X2088_c9
6480.v4.c25
6952.v1.c20
6811.v7.c18
0815.v3.c3
B
B (T/F)
B (T/F)
B (T/F)
C
C (T/F)
C (T/F)
C (T/F)
C (T/F)
BC
CRF02_AG
G
CD
CD
CD
ACD
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
10.817
6.916
7.674
4.305
2.136
1.183
11.145
1.262
6.763
9.645
16.982
10.453
1.476
1.666
5.080
1.399
WEAU_d15_410_5017
HIV-16845-2.22
620345.c01
0815.v3.c3
6480.v4.c25
6952.v1.c20
6811.v7.c18
X2088_c9
3016.v5.c45
191821_E6_1
246F C1G
7030102001E5(Rev-)
CNE30
Ce1086_B2
ZM214M.PL15
PVO.4
TRO.11
RHPA4259.7
THRO4156.18
1054_07_TC4_1499
6244_13_B5_4576
62357_14_D3_4589
B (T/F)
C
CRF01_AE
ACD
CD
CD
CD
G
D
D (T/F)
C (T/F)
C (T/F)
BC
C (T/F)
C
B
B
B
B
B (T/F)
B (T/F)
B (T/F)
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
⬎50
19.772
18.415
14.409
7.406
8.097
11.544
24.897
30.361
1.571
0.976
3.500
29.579
41.902
4.069
18.933
8.239
35.584
7.092
20.071
36.094
28.640
17.254
a
ID, identification.
T/F, transmitted/founder virus.
c
The PG9 data are historical data for this 117-virus panel bridged for the current
experiment with a set of representative viruses. For PG9-aplaviroc, the median IC50
titer in TZM-bl cells is 5.921 ␮g/ml, and the mean ⫾ standard deviation is 6.181 ⫾
4.703 ␮g/ml.
b
a
ruses with IC80s of ⬍50 ␮g/ml; for 22 of the pseudovirus strains
neutralized by the PG9-aplaviroc conjugate, the IC80 of the parent
antibody, PG9, was above 50 ␮g/ml (Table 4). Unlike the parent
antibody, the conjugate neutralized all clade B and C viruses, including transmitted/founder viruses. Clade C is predominant in Southern
and East Africa, India, and Nepal and is responsible for about half of
worldwide infections. Clade B is the major cause of infection in the
Americas, Europe, Japan, and Australia. Thus, conjugation to aplaviroc significantly improved the breadth of neutralization ability.
The PG9-aplaviroc conjugate demonstrated a dramatic improvement over the parent antibody in the maximum percentage
of inhibition (MPI) across the 117-virus panel (Table 5; see also
the supplemental material). PG9 is sensitive to the glycosylation
profile of a virus and cannot completely neutralize certain viral
strains even at high antibody concentrations (34). The PG9-aplaviroc conjugate did not display this glycan sensitivity and was
found to be more potent than native PG9 when MPIs were compared. PG9 and PG9-aplaviroc neutralized 40 and 84 of the 117
viral isolates tested at MPIs of ⬎99.5%, respectively.
DISCUSSION
The success of HAART is predicated on the fact that although
HIV-1 is a hypermutating virus, a therapy consisting of a combination of inhibitors targeting three viral enzymes—reverse transcriptase, protease, and integrase—presents a stringent barrier to
viral replication. HAART often reduces viral replication to undetectable levels in patients. HAART does not, however, present an
insurmountable barrier to viral replication, and drug resistance
can evolve rapidly when patient compliance is poor. Two key factors that contribute to drug compliance failure are frequency of
administration, which has been reduced to once daily for some
HAART regimens, and adverse side effects, often driven by meta-
4990
jvi.asm.org
ID, identification.
T/F, transmitted/founder virus.
c
The PG9 data are historical data for this 117-virus panel bridged for the current
experiment with a set of representative viruses. For PG9-aplaviroc, the median IC80 titer in
TZM-bl cells is 17.834 ␮g/ml, and the mean ⫾ standard deviation is 17.655 ⫾ 12.160
␮g/ml.
b
bolic toxicity. Both of these limitations might be addressed by the
development of a HAART equivalent based on protein drugs. For
example, antibodies have long in vivo half-lives. Native human
IgG1 has a t1/2 of 23 days in humans, and engineered variants of
TABLE 5 Summary of MPIsa for PG9 and PG9-aplaviroc
Clade
No. of
viruses
tested
A
A (T/F)
B
B (T/F)
C
C (T/F)
D and D (T/F)
G
AC
AG
AE
AE (T/F)
ACD
BC
CD
8
3
12
9
16
15
5
7
4
9
10
4
2
8
5
b
a
b
No. of viruses
neutralized with an
MPI of 100%
Avg MPI
PG9
PG9-aplaviroc
PG9
PG9-aplaviroc
3
3
1
0
7
10
1
1
1
3
5
0
0
5
0
6
3
4
2
12
13
3
4
3
7
7
2
0
6
2
97
100
77
63
88
76
83
85
98
92
96
93
29
89
47
99
100
95
94
99
97
99
98
100
97
99
100
84
98
97
MPIs of ⬎99.5% were rounded up to 100% by the analysis software.
T/F, transmitted/founder virus.
Journal of Virology
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Virus IDa
Potent, Broadly Active Bispecific Anti-HIV Antibodies
May 2013 Volume 87 Number 9
roc derivatives that are amenable to linkage to proteins or longlived carriers have not yet been described.
We prepared aplaviroc conjugates with the broadly neutralizing antibodies b12, 2G12, PG9, PG16, and CD4-IgG. The
incorporation of aplaviroc significantly improved the neutralization abilities of BNmAbs for strains resistant to or weakly
neutralized by the parent antibodies. The antiviral activity of
PG9-aplaviroc was analyzed against a panel of 117 different
viral strains. The conjugate neutralized the activities of 100% of
viral strains with IC50s lower than 50 ␮g/ml. To the best of our
knowledge, none of the currently known BNmAbs neutralize
all of these 117 cross-clade pseudoviruses with IC50s lower than
50 ␮g/ml. Notably, compared to parent PG9-aplaviroc had
lower IC50s against 13 out of 32 tested transmitted/founder
viruses of different clades and was more potent than any of the
parent molecules alone against 10 of these viruses (see the supplemental material). PG9-aplaviroc also displayed MPIs significantly improved over those of the parent antibody.
The median IC50 of the PG9-aplaviroc conjugate against the
panel was 2.76 nM, whereas that of the parent antibody, PG9, was
0.77 nM. On a panel of 76 of 117 viruses, PG9-aplaviroc had
higher IC50s than the naked PG9 antibody (see section S5 in the
supplemental material), with median IC50s of 0.674 nM for PG9
and 1.737 nM for PG9-aplaviroc. Notably, aplaviroc itself was not
very potent on this set of the viruses, with a median IC50 of 4.6 nM.
We hypothesize that during the neutralization assay there is competition between binding to CCR5 and binding to the HIV-1 envelope, and thus, the conjugate is not always used in the most
efficient neutralization pathway. Since these two mechanisms of
neutralization are in competition when the conjugate is assayed
against viruses that are very sensitive to PG9 neutralization, some
reduction in potency against these isolates is expected.
Combining potent molecules that prevent HIV entry through
different mechanisms into a single multifunctional molecule may
create an insurmountable evolutionary challenge limiting the development of resistance. This hypothesis is supported by a recent
report by Zhou et al. showing that HIV escape mutants selected by
exposure to entry inhibitors were much more sensitive to BNmAbs than the original virus (44). The chemical approach to the
synthesis of multispecific BNmAbs described here will allow ready
access to various combinations of neutralizing antibodies and
host-protecting small-molecule drugs that bind to the receptors
crucial for HIV entry (CCR5, CXCR4, CD4). Intelligent design of
the linker architecture will allow the preparation of tri- and tetraspecific antibody conjugates.
ACKNOWLEDGMENTS
This work was supported by NIH grants AI095038 (C.F.B.), AI33292
(D.R.B.), AI100663 (D.R.B.), and IAVI (D.R.B.).
We thank B. Hahn, F. McCutchan, G. Shaw, D. Montefiori, M. Thomson, J. Overbaugh, R. Swanstrom, L. Morris, J. Kim, L. Zhang, D. Ellensberger, and C. Williamson for contributing the HIV-1 envelope plasmids
used in the extended neutralization panel.
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SUPPORTING INFORMATION FOR:
Antibody Conjugation Approach Enhances Breadth and Potency of
Neutralization of anti-HIV-1 Antibodies and CD4-IgG
running title: Potent, Broadly Active Bispecific anti-HIV Antibodies
Julia Gavrilyuk,1 Hitoshi Ban,1,2 Hisatoshi Uehara,1,3 Shannon Sirk,1 Karen Saye,4
Angelica Cuevas,4 Elise Zablowsky,5 Avinash Oza,5 Michael S. Seaman,5 Dennis
R. Burton,4 and Carlos F. Barbas, III*1
1
Departments of Chemistry and Molecular Biology and the Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla, California, USA;
2
Dainippon Sumitomo Pharma, Osaka, Japan;
3
Mitsubishi Chemical Group
Science and Technology Research Center, Yokohama, Japan; 4Department of
Immunology and Microbial Science and IAVI Neutralizing Antibody Center and
Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The
Scripps Research Institute, La Jolla, California, USA; 5Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts, USA
Corresponding Author: Carlos F. Barbas, III Departments of Chemistry and
Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps
Research Institute, La Jolla, CA 92037, carlos@scripps.edu
1 Table of contents.
1. MALDI-TOF characterization of antibody conjugates. ....................................... 3 2. Tryptic digest and MS/MS characterization of antibody conjugates. .......... 9 3. Optimization studies for the modification of model antibody
(Trastuzumab)....................................................................................................................... 11 4. Additional Neutralization Experiments.................................................................. 16 5. Neutralization Results for 117 Pseudovirus Panel Screen............................ 17 6. PG9 and PG9-aplaviroc MPI full summary.......................................................... 32 2 1. MALDI-TOF characterization of antibody conjugates.
Trastuzumab-aplaviroc:
Mav (trastuzumab) = 148348
Mav (trastuzumab-apl) = 149243
3 IgG b12-aplaviroc:
Mav (b12) = 151960
Mav (b12-apl) = 152347
4 IgG 2G12-aplaviroc:
Mav (2G12) = 149397
Mav (2G12-apl) = 151464
5 IgG PG9-aplaviroc:
Mav (PG9) = 155168
Mav (PG9-apl) = 156408
6 IgG PG16-aplaviroc:
Mav (PG16) = 154644
Mav (PG16-apl) = 157377
7 CD4-IgG-aplaviroc:
MavMALDI-TOF
(CD4-IgG) = 177125
Mav (CD4-IgG-apl) = 178900
Method: BSAK
Laser : 2300
Mode: Linear
Scans Averaged: 140
Accelerating Voltage: 25000
Pressure: 4.50e-07
Grid Voltage: 94.000 %
Low Mass Gate: 4000.0
This File # 1 : D:\DATA\ROUTINE\2011\NOV\110911\SM6008.MS
Guide Wire Voltage: 0.300 %
Timed Ion Selector: 26.9 OFF
Comment:
Delay: 50 ON
Negative Ions: OFF
Sample: 93
Collected: 11/10/11 8:10 AM
Original Filename: d:\data\routine\2011\nov\110911\6008s3.ms
Savitsky-Golay Order = 2 Points = 19
1000
800
Counts
600
400
200
150000
160000
170000
180000
Mass (m/z)
190000
200000
210000
8 2. Tryptic digest and MS/MS characterization of antibody
conjugates.
Tryptic digest procedure.
Purified antibody samples (100 µl, 1 mg/ml) were exchanged into 6 M guanidine,
0.1 M Tris, pH 8.0 using 0.5-ml Zeba spin desalting columns (7k MWCC) as per
the manufacturer’s instructions. To each sample, 1 M DTT (Fisher) was added to
a final concentration of 20 mM. The samples were incubated for one hour at
37°C with gentle shaking, and a 1 M solution of iodoacetamide (Fisher) was
added to each sample to a final concentration of 40 mM.
Samples were
incubated at room temperature in the dark for 40 min. The alkylation reactions
were quenched by adding 1 M DTT to a 40 mM final concentration. Sample
buffer was then exchanged to trypsin digestion buffer (50 mM Tris, pH 7.5, 5 mM
CaCl2) using 0.5-ml Zeba spin desalting columns (7k MWCC). Trypsin Gold (0.5
mg/ml, Pierce) was dissolved in 50 mM acetic acid. Trypsin solution was added
to each sample at a 1:20 ratio (enzyme:protein, weight/weight), and samples
were incubated at 37ºC with shaking at 600 rpm for approximately 18 hours.
Digestion was stopped by addition of TFA to approximately 0.1%. Samples (20 µg protein injected) were analyzed using an Agilent 6510 Q-TOF
mass spectrometer with Zorbax SB-C18 narrow-bore, 2.1x150 mm, 3.5-µm
column (Agilent). HPLC parameters were as follows: flow rate 0.2 ml/min,
gradient from 0 to 40% mobile phase B over 80 min followed by a gradient to 0%
B from 80 to 90 min. Mobile phase A was 0.05% TFA (v/v) in HPLC-grade H2O,
2% acetonitrile (v/v), and mobile phase B was 0.04% TFA (v/v) in 90%
acetonitrile (v/v). MS data was collected for 200-2500 m/z and 100-2000m/z,
positive polarity, gas temperature 325 °C, nebulizer 30 psi, capillary voltage 3500
V. Data was analyzed using MassHunter Software (Agilent) and GPMAW
Version 8.20 Software (ChemSW).
Analysis summary.
Analysis was done using published antibody sequences.
Non-modified antibody samples were also digested and analyzed as controls
(data not shown).
9 MassHunter Bioconfirm workflow was used with the following specifications:
“Find by molecular feature” was run with the Isotope Model “Unbiased” and
limiting charged state to a maximum of 5. Allowed ion species included positive
ions +H, +Na; negative ions –H; neutral loss H2O. Other parameters were left at
default values.
Aplaviroc-FBDP modification was added to the chemical modifications library and
systematically applied to sequence and searched for the target modified peptide
fragments. All cysteine residues were modified with iodoacetamide.
Structures of the found aplaviroc-modified fragments were confirmed by MSMS
fragmentation pattern, reference MSMS fragmentation ions was calculated using
GPMAW.
Table1. Summary of the identified modification positions.
!"#$%&'(
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-7449=>?#@A9B>#CDE
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::*36<89I<364
-3I9=>?#@A9B>#CDE
-74<9=>?#@A9B>#CDE
76:4FG47
<64:FG4*
3M:3
3M:3
3M:3
3 FF/-+1!-2
33;*64H389::*3655489I<3647<
3 N//L+JO1K1J0PF,.P+P-!/Q7;<*6;*:89:H736H3*89:3;;6H53
3 1L!,1JJK..++O-R2
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::*36<89I<364
:3;H
:*7:6389H5*65
-*4*=>?#@A9B>#CDE
-5**=>?#@A9B>#CDE
-:7H9=SCM>%9B>#CDE
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:6;4FG4*
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RLH
RLH
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3 1L!,1JJK..++O-R2
33;*64H389::*3655489I<3647<
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::*365589I<364
:*7:6389H5*65
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76*4FG4*
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RL:<
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3 F-Q
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:::I64789I*564
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-5H=>?#@A9B>#CDE
-*:7=>?#@A9B>#CDE
-*7<=>?#@A9B>#CDE
3654FG43
76:4FG47
:634FG43
10 3. Optimization studies for the modification of model antibody
(Trastuzumab).
Optimization study was performed with trastuzumab as model IgG in order to
identify optimal procedure for antibody modification, assess reproducibility and
protein recovery.
General protocol for the two-step procedure.
Step 1: To the 200 µL of trastuzumab solution (1.5 mg/ml, 0.1M Na2HPO4
solution pH 8.0) was added FBDP solution in CH3CN (2 µL of 20 mM solution).
Reaction mixture was vortexed gently and allowed to react for 30 min. Reaction
turned yellow. The reaction was run through the gel filtration spin column to
remove excess FBDP and exchange buffer to 0.1M phosphate buffer pH 6.0.
Results summarized in Table 2.
Step 2: To the solution of FBDP modified antibody from step 1 were added 4 µL
of 20 mM solution of aplaviroc-oxyamine, reaction was vortexed and allowed to
react at 4oC for 18h. The resulting conjugate was purified using gel filtration spin
column, 20 µL stacker used. Results summarized in Table 3.
Protein concentration in the resulting solution was determined using two
independent methods. One method based on the absorbance at 280 nm
(Nanodrop) with the correction for the absorbance of diazene product at 340 nm,
correction factor of 0.436. Trastuzumab extinction coefficient was calculated
using
trastuzumab
sequence
and
ExPasy
ProtParam
tool
(http://web.expasy.org/cgi-bin/protparam/protparam). Another was based on the
11 Bradford test for protein concentration, using unmodified trastuzumab solution of
known concentration as standard.
Table 2. Summary of the first step experiments in two-step antibody modification
procedure.
#
Her
conc.,
µM
FBDP
eq
Precipitation
observed
Column for
gel
filtration
Recovery,
%
Comments
1
6.7
10
No
Bio-Rad
69
Phosphate buffer pH 7.4
2
6.7
10
No
Bio-Rad
66
Phosphate buffer pH 8.0
3
6.7
10
No
Bio-Rad
69
Phosphate buffer pH 7.4 +
0.1M urea
4
100
10
No
Bio-Rad
98
Phosphate buffer pH 8.0
5
10
10
No
Bio-Rad
13
Phosphate buffer pH 8.0
6
10
10
No
Bio-Rad
35
Phosphate buffer pH 8.0
7
10
10
No
Zeba
65
Phosphate buffer pH 8.0
8
10
10
No
Zeba
60
Phosphate buffer pH 8.0
9
17
10
No
Zeba
99
Phosphate buffer pH 8.0
10
10
10
No
Bio-Rad
83
Phosphate buffer pH 8.0
11
10
10
No
Zeba
99
Phosphate buffer pH 8.0
12
10
10
No
Bio-Rad
77
Phosphate buffer pH 8.0
13
10
10
No
Zeba
99
Phosphate buffer pH 8.0
14
10
10
No
Bio-Rad
84
Phosphate buffer pH 8.0
15
10
10
No
Zeba
99
Phosphate buffer pH 8.0
Table 3. Summary of the second step experiments in two-step antibody
modification procedure.
#
AplavirocONH2 eq.
Column for
gel filtration
Recovery, %
Average # of
modifications/Ab
Comments
1
29
Bio-Rad
50
0.96
o/n, 4 C
2
29
Bio-Rad
54
0.69
o/n, 4 C
3
29
Bio-Rad
56
0.85
o/n, 4 C
4
20
Bio-Rad
99
1.56
o/n, 4 C
o
o
o
o
12 o
5
20
Bio-Rad
40
-
o/n, 4 C
6
20
Bio-Rad
30
-
o/n, 4 C
7
20
Zeba
87
2.25
o/n, 4 C
8
20
Zeba
73
2.5
o/n, 4 C
9
10
Zeba
99
2.13
o/n, 4 C
10
20
Bio-Rad
58
1.54
o/n, 4 C
11
20
Zeba
71
2.07
o/n, 4 C
12
20
Bio-Rad
62
-
o/n, 4 C
13
20
Zeba
71
-
o/n, 4C
14
20
Bio-Rad
57
-
o/n, 4C
15
20
Zeba
71
2.03
o/n, 4C
o
o
o
o
o
o
o
General protocol for the one-step antibody modification procedure.
In 150 µL eppendorf tube were combined 1.6 µL of 100 mM acid solution in
CH3CN and 4 µL of 20 mM aplaviroc-oxyamine followed by 4 µL of 20 mM FBDP
in CH3CN and mixed, allowed to preform the oxime at rt for 30 min. Then, 200
µL of trastuzumab solution (1.5 mg/ml, 0.1M Na2HPO4 buffer pH 8.0) were
added, vortexed gently and allowed to react for 30 min at rt. Reaction mixture
turned yellow. Reaction mixture was run through the gel filtration spin column to
remove excess of the reagents, 10 µL stacker used. Results summarized in
Tables 4 and 5.
Protein concentration in the resulting solution was determined using two
independent methods. One method based on the absorbance at 280 nm
(Nanodrop) with the correction for the absorbance of diazene product at 340 nm,
correction factor of 0.436. Trastuzumab extinction coefficient was calculated
using
trastuzumab
sequence
and
ExPasy
ProtParam
tool
(http://web.expasy.org/cgi-bin/protparam/protparam). Another was based on the
Bradford test for protein concentration, using unmodified trastuzumab solution of
known concentration as standard.
13 Table 4. Summary of the optimization experiments in one-step antibody
modification procedure at pH 8.0.
#
Acid
Column for gel
Additive
filtration
Recovery, %
#
Precipitation
modifications/Ab
observed
1
-
Bio-Rad
64
0.36
no
2
AcOH
Bio-Rad
68
0.3
no
3
HCl
Bio-Rad
65
0
no
4
TFA
Zeba
97
0.47
yes
5
TFA
Zeba
99
0.28
yes
6
TFA
Zeba
45
0.39
yes
7
HCl
Zeba
99
0.51
yes
8
AcOH
Zeba
99
0.21
yes
9
TFA
Zeba
95
0.52
yes
10
HCl
Zeba
99
0.41
yes
11
AcOH
Zeba
99
0.14
yes
12
TFA
Zeba
99
0.31
yes
Table 5. Summary of the optimization experiments in one-step antibody
modification procedure at pH 9.0.
#
Acid additive
Column
for
% recovery
# modifications/Ab
gel filtration
1
TFA
Zeba
82
0.44
2
H2SO4 (aq)
Zeba
99
-
3
H3PO4 (conc).
Zeba
95
0.46
4
TFA
Zeba
91
0.56
5
HCl
Zeba
99
1.95
6
H2SO4
Zeba
86
0.39
7
H3PO4
Zeba
99
1
14 8
HCl
Bio-Rad
71
0.7
9
H3PO4
Bio-Rad
75
0.9
10
HCl
Bio-Rad
75
0
11
H3PO4
Bio-Rad
57
0.2
12
HCl
Bio-Rad
71
0.6
13
H3PO4
Bio-Rad
75
1
14
HCl
Zeba
76
0.3
15
H3PO4
Zeba
99
1
16
HCl
Zeba
99
0.4
17
HCl
Zeba
97
0.82
18
H3PO4
Zeba
87
1.54
19
H3PO4
Zeba
86
0.84
15 4. Additional Neutralization Experiments.
Neutralization of the CCR5-tropic HIV-1 JR-FL and YU2 by 1:1 molar
mixture PG9 and Aplaviroc.
Neutralization of the CXCR4-tropic HIV-1 HXB2 virus.
16 5.Neutralization Results for 117 Pseudovirus Panel Screen.
IC50s for the full panel.
17 Virus ID
6535.3
QH0692.42
SC422661.8
PVO.4
TRO.11
AC10.0.29
RHPA4259.7
THRO4156.18
REJO4541.67
TRJO4551.58
WITO4160.33
CAAN5342.A2
Median
Mean
STD
WEAU_d15_410_5017
1006_11_C3_1601
1054_07_TC4_1499
1056_10_TA11_1826
1012_11_TC21_3257
6240_08_TA5_4622
6244_13_B5_4576
62357_14_D3_4589
SC05_8C11_2344
Median
Mean
STD
Du156.12
Du172.17
Du422.1
ZM197M.PB7
ZM214M.PL15
ZM233M.PB6
ZM249M.PL1
ZM53M.PB12
ZM109F.PB4
ZM135M.PL10a
CAP45.2.00.G3
CAP210.2.00.E8
HIV-001428-2.42
HIV-0013095-2.11
HIV-16055-2.3
HIV-16845-2.22
Median
Mean
STD
Ce1086_B2
Ce0393_C3
Ce1176_A3
Ce2010_F5
Ce0682_E4
Ce1172_H1
Ce2060_G9
Ce703010054_2A2
BF1266.431a
246F C1G
249M B10
ZM247v1(Rev-)
7030102001E5(Rev-)
1394C9G1(Rev-)
Ce704809221_1B3
Median
Mean
STD
CNE19
CNE20
CNE21
CNE17
CNE30
CNE52
CNE53
CNE58
Clade*
B
B
B
B
B
B
B
B
B
B
B
B
IC50 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9**
1.021
1.173
17.438
>333
7.309
3.687
0.805
70.427
19.277
288.313
25.034
0.78
2.843
150.8
11.205
163.167
21.009
0.08
25.481
3.353
4.593
0.067
6.255
30.687
9.257
3.687
11.856
64.776
9.319
96.053
PG9/aplaviroc
3.294
72.112
15.193
4.773
43.624
1.277
13.569
32.376
0.134
7.468
0.078
31.339
10.518
18.770
22.181
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
4.078
0.366
>50
6.339
0.172
1.747
>50
>50
0.892
1.320
2.266
2.450
1.087
0.466
6.916
4.513
0.470
2.998
7.674
4.305
1.385
2.998
3.313
2.724
100.000
4.086
7.873
6.238
21.247
14.962
9.738
4.156
5.663
7.873
19.329
30.770
27.187
2.44
>333
42.26
1.147
11.647
>333
>333
5.947
8.797
15.105
16.337
7.250
3.109
46.107
30.084
3.132
19.989
51.160
28.700
9.233
19.989
22.085
18.163
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
0.026
0.606
0.614
0.899
>50
0.010
0.166
0.069
0.256
20.559
0.007
0.040
0.101
0.010
0.021
4.398
0.101
1.852
5.293
0.088
0.581
0.512
0.442
2.136
0.003
0.110
0.112
0.309
5.023
0.003
0.261
0.003
0.039
0.022
4.444
0.186
0.880
1.594
17.108
10.435
10.456
2.343
2.419
0.847
0.817
0.656
1.325
5.045
0.693
15.878
4.599
0.947
1.781
5.539
2.381
5.056
5.482
0.173
4.04
4.093
5.993
>333
0.067
1.107
0.46
1.707
137.06
0.0467
0.267
0.673
0.067
0.14
29.32
0.673
12.348
35.287
0.588
3.871
3.412
2.947
14.241
0.023
0.734
0.745
2.061
33.485
0.017
1.737
0.017
0.263
0.145
29.624
1.241
5.869
10.629
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
>50
0.020
0.014
>50
0.196
0.070
0.045
0.022
0.026
>50
0.095
0.112
>50
0.029
0.037
0.037
0.061
0.061
1.183
0.024
0.034
11.145
0.208
0.106
0.104
0.014
0.080
1.262
0.129
0.138
6.763
0.043
0.201
0.129
1.429
1.429
1.145
0.330
14.886
19.793
1.901
6.919
4.183
0.394
1.471
1.734
0.699
0.891
5.871
2.306
12.650
1.901
5.012
5.012
>333
0.133
0.093
>333
1.307
0.467
0.3
0.147
0.173
>333
0.633
0.747
>333
0.193
0.247
0.247
0.404
0.404
7.888
0.157
0.224
74.297
1.384
0.706
0.695
0.095
0.532
8.414
0.863
0.918
45.087
0.286
1.339
0.863
9.526
9.526
BC
BC
BC
BC
BC
BC
BC
BC
0.024
0.052
0.038
0.210
>50
0.029
0.131
0.038
0.038
0.075
0.070
0.055
0.182
0.112
0.332
9.645
0.176
0.700
0.229
0.205
1.429
3.326
2.444
1.694
0.748
5.516
11.919
>100
18.158
20.777
5.516
8.751
8.239
0.16
0.347
0.253
1.4
>333
0.193
0.873
0.253
0.253
0.497
0.466
0.364
1.216
0.745
2.213
64.297
1.176
4.666
1.524
1.370
9.525
22.171
A
A
A
A
A
A
A
A
0.005
0.027
1.484
0.025
0.029
0.023
0.026
1.693
0.0265
0.414
0.727
0.110
0.013
1.470
0.020
0.140
0.058
0.035
1.866
0.084
0.464
0.752
0.202
13.939
3.681
3.424
10.726
37.578
2.390
17.280
7.2035
11.153
12.283
0.033
0.18
9.893
0.167
0.193
0.153
0.173
11.287
0.1765
2.760
4.847
0.734
0.090
9.798
0.134
0.935
0.387
0.235
12.437
0.560
3.094
5.011
Median
Mean
STD
MS208.A1
Q23.17
Q461.e2
Q769.d22
Q259.d2.17
Q842.d12
0330.v4.c3
0260.v5.c36
Median
Mean
STD
IC50 Titer in TZM-bl cells, µg/ml
PG9**
PG9/aplaviroc
0.176
0.494
>50
10.817
0.553
2.279
10.564
0.716
43.247
6.544
0.117
0.192
22.620
2.035
24.475
4.856
0.012
0.020
0.503
1.120
0.010
0.012
4.603
4.701
0.553
1.578
9.716
2.815
14.408
3.327
18 IC50s continued:
191955_A11
191084 B7-19
9004SS_A3_4
Median
Mean
STD
A (T/F)
A (T/F)
A (T/F)
0.053
0.043
0.088
0.053
0.061
0.024
0.017
0.091
0.130
0.091
0.079
0.058
0.032
4.471
5.825
4.471
3.443
3.030
0.353
0.287
0.587
0.353
0.409
0.158
0.112
0.605
0.868
0.605
0.528
0.384
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
0.037
0.101
0.293
0.005
>50
0.393
0.023
0.049
0.233
0.075
0.142
0.146
0.089
0.213
0.541
0.001
16.982
1.484
0.048
0.215
0.531
0.215
2.234
5.549
16.777
9.155
4.779
14.339
22.188
9.074
6.700
19.148
1.075
9.155
11.471
7.044
0.247
0.673
1.953
0.033
>333
2.62
0.153
0.327
1.553
0.5
0.945
0.971
0.594
1.423
3.608
0.007
113.210
9.893
0.319
1.434
3.540
1.434
14.892
36.994
620345.c01
C1080.c03
R2184.c04
R1166.c01
R3265.c06
C2101.c01
C3347.c11
C4118.c09
CNE5
BJOX009000.02.4
Median
Mean
STD
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
1.333
0.004
0.255
0.736
0.163
0.034
0.032
0.068
0.018
1.725
0.116
0.437
0.622
2.278
0.006
0.666
1.108
0.423
0.032
0.176
0.071
0.020
2.957
0.300
0.774
1.045
6.946
25.834
5.517
4.644
3.334
1.832
1.756
0.603
1.186
3.915
3.625
5.557
7.405
8.887
0.0267
1.7
4.907
1.087
0.227
0.213
0.453
0.12
11.5
0.770
2.912
4.149
15.187
0.039
4.443
7.389
2.818
0.213
1.176
0.476
0.134
19.714
1.997
5.159
6.969
BJOX015000.11.5
BJOX010000.06.2
BJOX025000.01.1
BJOX028000.10.3
Median
Mean
STD
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
0.322
0.154
0.144
1.100
0.238
0.430
0.454
0.748
0.603
0.309
0.597
0.600
0.564
0.184
2.991
1.529
0.436
0.372
0.983
1.332
1.227
2.147
1.027
0.96
7.333
1.587
2.867
3.027
4.986
4.023
2.061
3.977
4.000
3.762
1.225
X1193_c1
P0402_c2_11
X1254_c3
X2088_c9
X2131_C1_B5
P1981_C5_3
X1632_S2_B10
Median
Mean
STD
G
G
G
G
G
G
G
0.105
0.296
0.065
>50
0.084
0.258
0.107
0.106
0.153
0.098
0.355
0.317
0.311
10.453
0.511
1.062
0.681
0.511
1.956
3.757
2.076
1.341
3.047
12.647
3.621
25.451
6.005
3.621
7.741
8.687
0.7
1.973
0.433
>333
0.56
1.72
0.713
0.7065
1.017
0.656
2.364
2.112
2.073
69.687
3.406
7.082
4.538
3.406
13.037
25.044
3016.v5.c45
A07412M1.vrc12
231965.c01
231966.c02
Median
Mean
STD
D
D
D
D
2.455
0.697
1.285
0.075
0.991
1.128
1.013
0.409
1.296
1.943
0.107
0.853
0.939
0.838
0.394
9.851
5.641
4.337
4.989
5.056
3.898
16.367
4.647
8.567
0.5
6.607
7.520
6.755
2.729
8.639
12.952
0.712
5.684
6.258
5.589
D (T/F)
3.600
1.285
1.622
1.411
0.313
0.409
0.813
0.778
0.188
4.337
4.082
4.017
24
8.567
10.816
9.410
2.084
2.729
5.423
5.187
CD
CD
CD
CD
CD
0.020
>50
>50
>50
0.668
0.344
0.344
0.458
0.003
1.476
1.666
5.080
0.587
1.476
1.763
1.973
4.230
1.378
1.642
6.954
0.573
1.642
2.955
2.622
0.133
>333
>333
>333
4.453
2.293
2.293
3.055
0.017
9.843
11.109
33.866
3.916
9.843
11.750
13.155
3301.v1.c24
6041.v3.c23
6540.v4.c1
6545.v4.c1
Median
Mean
STD
AC
AC
AC
AC
0.216
0.244
0.070
0.099
0.158
0.157
0.086
0.445
0.627
0.116
0.287
0.366
0.369
0.218
4.349
3.498
13.148
4.814
4.582
6.452
4.497
1.44
1.627
0.467
0.66
1.050
1.049
0.571
2.969
4.179
0.773
1.910
2.439
2.458
1.456
0815.v3.c3
3103.v3.c10
Median
Mean
STD
ACD
ACD
>50
28.004
28.004
28.004
1.399
27.728
14.563
14.563
18.617
1.532
63.328
32.430
32.430
43.696
>333
186.693
186.693
186.693
9.328
184.851
97.089
97.089
124.114
T257-31
928-28
263-8
T250-4
T251-18
T278-50
T255-34
211-9
235-47
Median
Mean
STD
191821_E6_1
Median(D+D(T/F))
Mean(D+D(T/F))
STD(D+D(T/F))
3817.v2.c59
6480.v4.c25
6952.v1.c20
6811.v7.c18
89-F1_2_25
Median
Mean
STD
Note: Calculation of Mean and Median omits any values with “>” sign.
19 Figure 1. Graphical representation of the IC50s for all samples. PG9 IC50s>333nM are omitted for clarity.
Statistical analysis for a set of pseudoviruses, where PG9 IC50<50 ug/ml.
The following Mean and Median IC50s were calculated by GraphPad Prism
analysis:
PG9 Median IC50 = 0.713nM, Mean IC50 =1.91nM
PG9/aplaviroc Median IC50 = 13.1nM, Mean IC50 = 6.79nM
Aplaviroc Median IC50 =4.35nM, Mean IC50 =7.56nM.
Mann Whitney test has shown that in this set of samples there is no statistically
significant difference between Median IC50 values of PG9 and PG9/aplaviroc (P
value 0.0547).
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC50 values of Aplaviroc and
PG9/aplaviroc (P value 0.0002).
20 Pseudoviruses where PG9-aplaviroc has improved IC50 relative to wildtype PG9.
Pseudoviruses neutralized by PG9 with IC50>50 µg/ml (>333nM) are grouped
separately.
Improved IC50s for PG9/apl vs PG9
Virus ID
PVO.4
TRO.11
RHPA4259.7
THRO4156.18
1056_10_TA11_1826
Du172.17
Du422.1
ZM197M.PB7
ZM233M.PB6
ZM249M.PL1
HIV-001428-2.42
Ce703010054_2A2
Q23.17
Q461.e2
Q769.d22
191955_A11
T250-4
C2101.c01
3016.v5.c45
191821_E6_1
3817.v2.c59
89-F1_2_25
3103.v3.c10
Median
Mean
STD
Clade*
B
B
B
B
B (T/F)
C
C
C
C
C
C
C (T/F)
A
A
A
A (T/F)
CRF02_AG
CRF01_AE
D
D (T/F)
CD
CD
ACD
IC50 Titer in TZM-bl cells, µg/ml
PG9**
PG9/aplaviroc
10.564
0.716
43.247
6.544
22.620
2.035
24.475
4.856
6.339
4.513
0.606
0.581
0.614
0.512
0.899
0.442
0.010
0.003
0.166
0.110
0.101
0.003
0.022
0.014
0.027
0.013
1.484
1.470
0.025
0.020
0.053
0.017
0.005
0.001
0.034
0.032
2.455
0.409
3.600
0.313
0.020
0.003
0.668
0.587
28.004
27.728
0.614
0.409
6.349
2.214
11.703
5.843
IC50 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9**
0.805
70.427
19.277
288.313
2.843
150.8
11.205
163.167
6.238
42.26
10.435
4.04
10.456
4.093
2.343
5.993
0.847
0.067
0.817
1.107
4.599
0.673
0.394
0.147
13.939
0.18
3.681
9.893
3.424
0.167
0.032
0.353
14.339
0.033
1.832
0.227
0.394
16.367
0.188
24
4.230
0.133
0.573
4.453
63.328
186.693
3.424
4.093
7.662
42.330
13.312
78.018
PG9/aplaviroc
4.773
43.624
13.569
32.376
30.084
3.871
3.412
2.947
0.023
0.734
0.017
0.095
0.090
9.798
0.134
0.112
0.007
0.213
2.729
2.084
0.017
3.916
184.851
2.729
14.760
38.953
Average improvement of 2.87 fold relative to PG9
Average decrease of potency relative to aplaviroc: 1.93 fold
Figure 2. Graphical representation of the improved IC50s. Pseudoviruses where PG9 has IC50>50 µg/ml
are grouped separately.
Statistical analysis for a set of pseudoviruses, where PG9/aplaviroc has
improved IC50s relative to PG9.
The following Mean and Median IC50s were calculated by GraphPad Prism
analysis:
PG9 Median IC50 = 4.09nM, Mean IC50 = 42.33nM
PG9/aplaviroc Median IC50 = 2.73nM, Mean IC50 = 14.76nM
Aplaviroc Median IC50 =3.42nM, Mean IC50 =7.66nM.
Mann Whitney test has shown that in this set of samples there is no statistically
significant difference between Median IC50 values of PG9 and PG9/aplaviroc (P
value 0.1533).
Mann Whitney test has shown that in this set of samples there is no statistically
significant difference between Median IC50 values of Aplaviroc and
PG9/aplaviroc (P value 0.3018).
21 Pseudoviruses neutralized by PG9 with IC50>50 µg/ml (>333nM):
Improved from IC50 >50 ug/ml
Virus ID
QH0692.42
1054_07_TC4_1499
6244_13_B5_4576
62357_14_D3_4589
ZM214M.PL15
Ce1086_B2
Ce2010_F5
246F C1G
7030102001E5(Rev-)
CNE30
T251-18
X2088_c9
6480.v4.c25
6952.v1.c20
6811.v7.c18
0815.v3.c3
Median
Mean
STD
Clade*
B
B (T/F)
B (T/F)
B (T/F)
C
C (T/F)
C (T/F)
C (T/F)
C (T/F)
BC
CRF02_AG
G
CD
CD
CD
ACD
IC50 Titer in TZM-bl cells, µg/ml
PG9**
PG9/aplaviroc
>50
10.817
>50
6.916
>50
7.674
>50
4.305
>50
2.136
>50
1.183
>50
11.145
>50
1.262
>50
6.763
>50
9.645
>50
16.982
>50
10.453
>50
1.476
>50
1.666
>50
5.080
>50
1.399
>50
5.921
>50
6.181
4.703
IC50 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9**
17.438
>333
7.873
>333
9.738
>333
4.156
>333
2.419
>333
1.145
>333
19.793
>333
1.734
>333
5.871
>333
11.919
>333
22.188
>333
12.647
>333
1.378
>333
1.642
>333
6.954
>333
1.532
>333
6.413
>333
8.027
>333
6.992
PG9/aplaviroc
72.112
46.107
51.160
28.700
14.241
7.888
74.297
8.414
45.087
64.297
113.210
69.687
9.843
11.109
33.866
9.328
39.476
41.209
31.354
TRO.11
Figure 3. Graphical representation of the IC50s of aplaviroc and PG9-aplaviroc in the panel of
pseudoviruses poorly neutralized by PG9 (IC50>50 µg/ml).
Statistical analysis for a set of pseudoviruses, where PG9 IC50>50ug/ml.
The following Mean and Median IC50s were calculated by GraphPad Prism
analysis:
PG9/aplaviroc Median IC50 = 39.48nM, Mean IC50 = 41.21nM
Aplaviroc Median IC50 =6.41nM, Mean IC50 =8.03nM.
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC50 values of Aplaviroc and
PG9/aplaviroc (P value 0.0003).
22 Pseudoviruses where PG9-aplaviroc shows decreased potency (IC50) relative to
wildtype PG9:
Decrease of potency of PG9/apl relative to PG9
Virus ID
6535.3
SC422661.8
AC10.0.29
REJO4541.67
TRJO4551.58
WITO4160.33
CAAN5342.A2
WEAU_d15_410_5017
1006_11_C3_1601
1012_11_TC21_3257
6240_08_TA5_4622
SC05_8C11_2344
Du156.12
ZM53M.PB12
ZM109F.PB4
CAP210.2.00.E8
HIV-0013095-2.11
HIV-16055-2.3
HIV-16845-2.22
Ce0393_C3
Ce1176_A3
Ce0682_E4
Ce1172_H1
Ce2060_G9
249M B10
ZM247v1(Rev-)
1394C9G1(Rev-)
Ce704809221_1B3
CNE19
CNE20
CNE21
CNE17
CNE52
CNE53
CNE58
MS208.A1
Q259.d2.17
Q842.d12
0330.v4.c3
0260.v5.c36
191084 B7-19
9004SS_A3_4
T257-31
928-28
263-8
T278-50
T255-34
211-9
235-47
620345.c01
C1080.c03
R2184.c04
R1166.c01
R3265.c06
C3347.c11
C4118.c09
CNE5
BJOX009000.02.4
BJOX015000.11.5
Clade*
B
B
B
B
B
B
B
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
C
C
C
C
C
C
C
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
BC
BC
BC
BC
BC
BC
BC
A
A
A
A
A
A (T/F)
A (T/F)
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE (T/F)
IC50 Titer in TZM-bl cells, µg/ml
PG9**
PG9/aplaviroc
0.176
0.494
0.553
2.279
0.117
0.192
0.012
0.020
0.503
1.120
0.010
0.012
4.603
4.701
4.078
1.087
0.366
0.466
0.172
0.470
1.747
2.998
0.892
1.385
0.026
0.088
0.069
0.112
0.256
0.309
0.040
0.261
0.010
0.039
0.021
0.022
4.398
4.444
0.020
0.024
0.014
0.034
0.196
0.208
0.070
0.106
0.045
0.104
0.095
0.129
0.112
0.138
0.029
0.043
0.037
0.201
0.024
0.055
0.052
0.182
0.038
0.112
0.210
0.332
0.029
0.176
0.131
0.700
0.038
0.229
0.005
0.110
0.029
0.140
0.023
0.058
0.026
0.035
1.693
1.866
0.043
0.091
0.088
0.130
0.037
0.089
0.101
0.213
0.293
0.541
0.393
1.484
0.023
0.048
0.049
0.215
0.233
0.531
1.333
2.278
0.004
0.006
0.255
0.666
0.736
1.108
0.163
0.423
0.032
0.176
0.068
0.071
0.018
0.020
1.725
2.957
0.322
0.748
IC50 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9**
1.021
1.173
7.309
3.687
25.034
0.78
21.009
0.08
25.481
3.353
4.593
0.067
6.255
30.687
>100
27.187
4.086
2.44
21.247
1.147
14.962
11.647
5.663
5.947
17.108
0.173
0.656
0.46
1.325
1.707
15.878
0.267
0.947
0.067
1.781
0.14
5.539
29.32
0.330
0.133
14.886
0.093
1.901
1.307
6.919
0.467
4.183
0.3
0.699
0.633
0.891
0.747
2.306
0.193
12.650
0.247
2.444
0.16
1.694
0.347
0.748
0.253
5.516
1.4
>100
0.193
18.158
0.873
20.777
0.253
0.202
0.033
10.726
0.193
37.578
0.153
2.390
0.173
17.280
11.287
4.471
0.287
5.825
0.587
16.777
0.247
9.155
0.673
4.779
1.953
9.074
2.62
6.700
0.153
19.148
0.327
1.075
1.553
6.946
8.887
25.834
0.0267
5.517
1.7
4.644
4.907
3.334
1.087
1.756
0.213
0.603
0.453
1.186
0.12
3.915
11.5
2.991
2.147
PG9/aplaviroc
3.294
15.193
1.277
0.134
7.468
0.078
31.339
7.250
3.109
3.132
19.989
9.233
0.588
0.745
2.061
1.737
0.263
0.145
29.624
0.157
0.224
1.384
0.706
0.695
0.863
0.918
0.286
1.339
0.364
1.216
0.745
2.213
1.176
4.666
1.524
0.734
0.935
0.387
0.235
12.437
0.605
0.868
0.594
1.423
3.608
9.893
0.319
1.434
3.540
15.187
0.039
4.443
7.389
2.818
1.176
0.476
0.134
19.714
4.986
Decreased potency (IC50) continued on the next page.
23 BJOX010000.06.2
BJOX025000.01.1
BJOX028000.10.3
X1193_c1
P0402_c2_11
X1254_c3
X2131_C1_B5
P1981_C5_3
X1632_S2_B10
A07412M1.vrc12
231965.c01
231966.c02
3301.v1.c24
6041.v3.c23
6540.v4.c1
6545.v4.c1
Median
Mean
STD
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
G
G
G
G
G
G
D
D
D
AC
AC
AC
AC
0.154
0.144
1.100
0.105
0.296
0.065
0.084
0.258
0.107
0.697
1.285
0.075
0.216
0.244
0.070
0.099
0.101
0.425
0.907
0.603
0.309
0.597
0.355
0.317
0.311
0.511
1.062
0.681
1.296
1.943
0.107
0.445
0.627
0.116
0.287
0.261
0.625
0.936
1.529
0.436
0.372
2.076
1.341
3.047
3.621
25.451
6.005
9.851
5.641
4.337
4.349
3.498
13.148
4.814
4.644
7.745
8.065
1.027
0.96
7.333
0.7
1.973
0.433
0.56
1.72
0.713
4.647
8.567
0.5
1.44
1.627
0.467
0.66
0.673
2.834
6.044
4.023
2.061
3.977
2.364
2.112
2.073
3.406
7.082
4.538
8.639
12.952
0.712
2.969
4.179
0.773
1.910
1.737
4.164
6.241
Average decrease of potency of PG9/apl relative to PG9: 1.47 fold
Average improvement of potency for PG9/apl relative to aplaviroc: 2.45 fold
Figure 4. Graphical representation of IC50s in the pseudovirus panel where PG9-aplaviroc has decreased
potency relative to PG9.
Statistical analysis for a set of pseudoviruses, where PG9/aplaviroc has
decreased potency relative to PG9.
The following Mean and Median IC50s were calculated by GraphPad Prism
analysis:
PG9 Median IC50 = 0.67nM, Mean IC50 = 2.73nM
PG9/aplaviroc Median IC50 = 1.74nM, Mean IC50 = 4.16nM
Aplaviroc Median IC50 =4.64nM, Mean IC50 =7.75nM.
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC50 values of PG9 and PG9/aplaviroc (P
value 0.0012).
Mann Whitney test has shown that in this set of samples there is no statistically
significant difference between Median IC50 values of Aplaviroc and
PG9/aplaviroc (P value <0.0001).
24 IC80s for the full panel.
Virus ID
6535.3
QH0692.42
SC422661.8
PVO.4
TRO.11
AC10.0.29
RHPA4259.7
THRO4156.18
REJO4541.67
TRJO4551.58
WITO4160.33
CAAN5342.A2
Median
Mean
STD
WEAU_d15_410_5017
1006_11_C3_1601
1054_07_TC4_1499
1056_10_TA11_1826
1012_11_TC21_3257
6240_08_TA5_4622
6244_13_B5_4576
62357_14_D3_4589
SC05_8C11_2344
Median
Mean
STD
Du156.12
Du172.17
Du422.1
ZM197M.PB7
ZM214M.PL15
ZM233M.PB6
ZM249M.PL1
ZM53M.PB12
ZM109F.PB4
ZM135M.PL10a
CAP45.2.00.G3
CAP210.2.00.E8
HIV-001428-2.42
HIV-0013095-2.11
HIV-16055-2.3
HIV-16845-2.22
Median
Mean
STD
Ce1086_B2
Ce0393_C3
Ce1176_A3
Ce2010_F5
Ce0682_E4
Ce1172_H1
Ce2060_G9
Ce703010054_2A2
BF1266.431a
246F C1G
249M B10
ZM247v1(Rev-)
7030102001E5(Rev-)
1394C9G1(Rev-)
Ce704809221_1B3
Median
Mean
STD
CNE19
CNE20
CNE21
CNE17
CNE30
CNE52
CNE53
CNE58
Clade*
B
B
B
B
B
B
B
B
B
B
B
B
IC80 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9*
12.517
4.18
98.769
>333
79.320
47.027
11.173
>333
100.000
>333
87.686
5.633
19.358
>333
61.850
>333
82.676
1.087
100.000
27.073
78.852
0.247
30.832
179.947
79.086
5.633
63.586
37.885
35.295
64.973
PG9/aplaviroc
9.933
>333
83.899
54.930
237.227
12.067
47.277
133.804
1.233
58.633
0.325
92.465
54.930
66.527
70.726
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
B (T/F)
>50
1.906
>50
38.259
0.605
6.333
>50
>50
4.283
4.283
10.277
15.796
19.772
2.119
36.094
23.408
4.402
10.408
28.640
17.254
6.784
17.254
16.542
11.592
>100
32.098
57.473
66.049
98.066
67.658
46.026
16.826
32.815
51.750
52.126
25.722
>333
12.707
>333
255.06
4.034
42.22
>333
>333
28.553
28.553
68.515
105.309
131.815
14.129
240.628
156.056
29.345
69.384
190.935
115.025
45.227
115.025
110.283
77.280
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
0.190
1.604
10.398
4.517
>50
0.026
1.410
0.221
6.689
>50
0.029
0.173
0.250
0.030
0.06
>50
0.221
1.969
3.261
0.342
2.746
2.824
2.214
18.933
0.030
0.409
0.509
2.457
21.958
0.006
3.241
0.027
0.170
0.110
18.415
1.362
4.649
7.622
93.800
62.373
52.130
13.654
40.579
3.294
4.246
6.009
10.570
21.910
15.920
78.371
28.984
7.069
8.758
31.308
18.915
29.936
28.166
1.267
10.693
69.32
30.113
>333
0.173
9.4
1.473
44.593
>333
0.193
1.153
1.667
0.2
0.4
>333
1.473
13.127
21.740
2.280
18.308
18.824
14.762
126.218
0.202
2.729
3.395
16.381
146.387
0.039
21.608
0.179
1.132
0.734
122.767
9.079
30.996
50.812
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
>50
0.055
0.044
>50
0.698
0.206
0.172
0.103
0.088
>50
0.290
0.323
>50
0.114
0.137
0.137
0.203
0.203
4.069
0.084
0.117
>50
1.507
0.530
0.297
0.089
0.192
3.500
0.508
0.565
29.579
0.155
1.233
0.519
3.030
3.030
4.462
2.152
87.151
>100
29.311
74.695
17.488
3.355
5.441
8.549
2.674
8.042
20.305
12.912
69.147
10.731
24.692
24.692
>333
0.367
0.293
>333
4.653
1.373
1.147
0.687
0.587
>333
1.933
2.153
>333
0.760
0.913
0.913
1.352
1.352
27.126
0.561
0.779
333.000
10.045
3.535
1.983
0.594
1.283
23.333
3.384
3.765
197.193
1.036
8.218
3.535
41.056
41.056
BC
BC
BC
BC
BC
BC
BC
BC
0.146
0.278
0.094
0.484
>50
0.081
0.60
0.122
0.146
0.257
0.206
0.245
0.637
0.392
1.171
41.902
0.821
3.285
0.732
0.776
6.148
14.478
17.066
8.431
5.283
29.677
39.067
100.000
60.248
51.858
34.372
38.954
31.571
0.973
1.853
0.627
3.227
>333
0.540
3.967
0.813
0.973
1.714
1.372
1.630
4.246
2.611
7.810
279.345
5.473
21.899
4.880
5.176
40.987
96.522
A
A
A
A
A
A
A
A
0.014
0.082
8.725
0.068
1.379
0.081
0.076
9.0
0.1
2.427
3.996
0.409
0.052
5.678
0.099
1.247
0.215
0.159
10.948
0.312
2.351
3.959
1.099
95.241
21.460
28.769
48.616
>100
8.747
97.906
28.769
43.120
39.519
0.093
0.547
58.167
0.453
9.193
0.540
0.507
59.953
0.543
16.182
26.642
2.728
0.347
37.854
0.661
8.314
1.434
1.059
72.986
2.081
15.673
26.390
Median
Mean
STD
MS208.A1
Q23.17
Q461.e2
Q769.d22
Q259.d2.17
Q842.d12
0330.v4.c3
0260.v5.c36
Median
Mean
STD
IC80 Titer in TZM-bl cells, µg/ml
PG9*
PG9/aplaviroc
0.627
1.490
>50
>50
7.054
12.585
>50
8.239
>50
35.584
0.845
1.810
>50
7.092
>50
20.071
0.163
0.185
4.061
8.795
0.037
0.049
26.992
13.870
0.845
8.239
5.683
9.979
9.746
10.609
25 IC80s continued:
191955_A11
191084 B7-19
9004SS_A3_4
Median
Mean
STD
A (T/F)
A (T/F)
A (T/F)
0.154
0.202
0.251
0.202
0.202
0.049
0.069
0.346
0.484
0.346
0.300
0.211
0.236
26.672
24.314
24.314
17.074
14.630
1.027
1.347
1.673
1.347
1.349
0.323
0.459
2.308
3.227
2.308
1.998
1.410
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
0.124
0.450
1.050
0.014
>50
3.192
0.134
0.243
1.201
0.347
0.801
1.062
0.353
1.587
1.784
0.005
>50
5.504
0.364
0.834
1.882
1.211
1.539
1.754
68.083
59.885
44.140
44.660
94.866
43.820
27.550
94.925
8.140
44.660
54.008
28.872
0.827
3.000
7.000
0.093
>333
21.280
0.893
1.620
8.007
2.310
5.340
7.081
2.353
10.577
11.894
0.034
>333
36.695
2.426
5.563
12.543
8.070
10.261
11.694
620345.c01
C1080.c03
R2184.c04
R1166.c01
R3265.c06
C2101.c01
C3347.c11
C4118.c09
CNE5
BJOX009000.02.4
Median
Mean
STD
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
>50
0.013
1.237
2.876
0.535
0.099
0.113
0.200
0.051
8.211
0.200
1.482
2.688
14.409
0.024
3.136
4.213
1.408
0.191
1.065
0.262
0.061
11.028
1.236
3.580
5.076
33.074
82.338
21.270
27.921
22.063
10.347
13.964
3.051
3.470
35.617
21.667
25.312
23.040
>333
0.087
8.247
19.173
3.567
0.660
0.753
1.333
0.340
54.740
1.333
9.878
17.922
96.062
0.162
20.908
28.090
9.384
1.272
7.098
1.748
0.409
73.518
8.241
23.865
33.841
BJOX015000.11.5
BJOX010000.06.2
BJOX025000.01.1
BJOX028000.10.3
Median
Mean
STD
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
1.489
0.740
0.335
21.500
1.115
6.016
10.334
5.628
2.804
2.721
4.000
3.402
3.788
1.358
45.477
19.761
18.749
17.776
19.255
25.441
13.382
9.927
4.933
2.233
143.333
7.430
40.107
68.892
37.518
18.695
18.140
26.667
22.681
25.255
9.056
X1193_c1
P0402_c2_11
X1254_c3
X2088_c9
X2131_C1_B5
P1981_C5_3
X1632_S2_B10
Median
Mean
STD
G
G
G
G
G
G
G
0.304
2.367
0.304
>50
0.286
2.512
0.934
0.619
1.118
1.054
1.745
1.821
1.557
30.361
2.383
5.532
5.046
2.383
6.921
10.464
16.344
19.745
17.921
55.550
19.084
71.099
96.596
19.745
42.334
32.323
2.027
15.780
2.027
>333
1.907
16.747
6.227
4.127
7.452
7.027
11.630
12.140
10.381
202.403
15.888
36.880
33.641
15.888
46.138
69.763
3016.v5.c45
A07412M1.vrc12
231965.c01
231966.c02
Median
Mean
STD
D
D
D
D
>50
7.963
47.969
0.257
7.963
18.730
25.613
1.571
6.715
12.029
0.708
4.143
5.256
5.236
2.193
49.448
49.438
14.785
32.112
28.966
24.197
>333
53.087
319.793
1.713
53.087
124.864
170.756
10.476
44.768
80.190
4.723
27.622
35.039
34.908
D (T/F)
>50
7.963
18.730
25.613
0.976
1.571
4.400
4.922
1.298
14.785
23.432
24.336
>333
53.087
124.864
170.756
6.510
10.476
29.333
32.813
3817.v2.c59
6480.v4.c25
6952.v1.c20
6811.v7.c18
89-F1_2_25
Median
Mean
STD
CD
CD
CD
CD
CD
0.062
>50
>50
>50
10.103
5.083
5.083
7.100
0.030
8.097
11.544
24.897
2.209
8.097
9.356
9.823
31.940
10.890
19.843
50.284
2.289
19.843
23.049
18.774
0.413
>333
>333
>333
67.353
33.883
33.883
47.334
0.202
53.983
76.958
165.983
14.728
53.983
62.371
65.486
3301.v1.c24
6041.v3.c23
6540.v4.c1
6545.v4.c1
Median
Mean
STD
AC
AC
AC
AC
0.756
3.639
0.253
0.351
0.554
1.250
1.608
2.568
3.224
0.694
1.082
1.825
1.892
1.200
24.002
13.488
55.967
25.819
24.911
29.819
18.260
5.040
24.260
1.687
2.340
3.690
8.332
10.718
17.120
21.490
4.627
7.210
12.165
12.612
8.002
0815.v3.c3
3103.v3.c10
Median
Mean
STD
ACD
ACD
>50
>50
>50
>50
7.406
50.000
28.703
28.703
30.119
7.363
100.000
53.682
53.682
65.504
>333
>333
>333
>333
49.373
>333
49.373
49.373
T257-31
928-28
263-8
T250-4
T251-18
T278-50
T255-34
211-9
235-47
Median
Mean
STD
191821_E6_1
Median(D+D(T/F))
Mean(D+D(T/F))
STD(D+D(T/F))
26 Figure 5. Graphical representation of the IC80s for all samples. PG9 IC80s>333nM are omitted for clarity.
Statistical analysis for all samples with PG9 IC80<50 ug/ml.
The following Mean and Median IC80s were calculated by GraphPad Prism
analysis:
PG9 Median IC80 = 1.98nM, Mean IC80 = 19.46nM
PG9/aplaviroc Median IC80 = 7.51nM, Mean IC80 = 17.00nM
Aplaviroc Median IC80 =27.55nM, Mean IC80 =37.22nM.
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC80 values of PG9 and PG9/aplaviroc (P
value 0.0161).
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC80 values of Aplaviroc and
PG9/aplaviroc (P value <0.0001).
27 Pseudoviruses where PG9-aplaviroc has improved IC80 relative to wildtype PG9.
Pseudoviruses neutralized by PG9 with IC80>50 µg/ml (>333nM) are grouped
separately.
Improved IC80s
Virus ID
1056_10_TA11_1826
Du172.17
Du422.1
ZM197M.PB7
ZM233M.PB6
ZM249M.PL1
HIV-001428-2.42
Ce703010054_2A2
Q23.17
Q461.e2
Q769.d22
191955_A11
T250-4
C2101.c01
3817.v2.c59
A07412M1.vrc12
6041.v3.c23
231965.c01
BJOX028000.10.3
CAAN5342.A2
ZM109F.PB4
P0402_c2_11
89-F1_2_25
Median
Mean
STD
Clade*
B (T/F)
C
C
C
C
C
C
C (T/F)
A
A
A
A (T/F)
CRF02_AG
CRF01_AE
CD
D
AC
D
CRF01_AE (T/F)
B
C
G
CD
IC80 Titer in TZM-bl cells, µg/ml
PG9*
PG9/aplaviroc
38.259
23.408
1.604
2.746
10.398
2.824
4.517
2.214
0.026
0.030
1.410
0.409
0.250
0.027
0.103
0.089
0.082
0.052
8.725
5.678
0.068
0.099
0.154
0.069
0.014
0.005
0.099
0.191
0.062
0.030
7.963
6.715
3.639
3.224
47.969
12.029
21.500
4.000
26.992
13.870
6.689
2.457
2.367
1.821
10.103
2.209
2.367
2.209
8.391
3.661
13.099
5.702
IC80 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9*
66.049
255.06
62.373
10.693
52.130
69.32
13.654
30.113
3.294
0.173
4.246
9.4
28.984
1.667
3.355
0.687
95.241
0.547
21.460
58.167
28.769
0.453
0.236
1.027
44.660
0.093
10.347
0.660
31.940
0.413
49.448
53.087
13.488
24.260
49.438
319.793
17.776
143.333
30.832
179.947
10.570
44.593
19.745
15.780
2.289
67.353
21.460
15.780
28.710
55.940
24.684
87.324
PG9/aplaviroc
156.056
18.308
18.824
14.762
0.202
2.729
0.179
0.594
0.347
37.854
0.661
0.459
0.034
1.272
0.202
44.768
21.490
80.190
26.667
92.465
16.381
12.140
14.728
14.728
24.405
38.016
Average improvement of 2.29 fold relative to PG9
Average decrease of potency relative to aplaviroc : 1.18 fold
Figure 6. Graphical representation of the improved IC80s. Pseudoviruses where PG9 has IC80>50 µg/ml
are grouped separately.
Statistical analysis for all samples with improved PG9/aplaviroc IC80s
relative to PG9. Sample set where PG9 has IC80> 50ug/ml is analyzed
separately.
The following Mean and Median IC80s were calculated by GraphPad Prism
analysis:
PG9 Median IC80 = 15.78nM, Mean IC80 = 55.94nM
PG9/aplaviroc Median IC80 = 14.73nM, Mean IC80 = 24.4nM
Aplaviroc Median IC80 =21.46nM, Mean IC80 =28.71nM.
Mann Whitney test has shown that in this set of samples there is no statistically
significant difference between Median IC80 values of PG9 and PG9/aplaviroc (P
value 0.3448).
28 Mann Whitney test has shown that in this set of samples there is no statistically
significant difference between Median IC80 values of Aplaviroc and
PG9/aplaviroc (P value 0.0826).
Pseudoviruses neutralized by PG9 with IC80>50 µg/ml (>333nM):
Virus ID
WEAU_d15_410_5017
HIV-16845-2.22
620345.c01
0815.v3.c3
6480.v4.c25
6952.v1.c20
6811.v7.c18
X2088_c9
3016.v5.c45
191821_E6_1
246F C1G
7030102001E5(Rev-)
CNE30
Ce1086_B2
ZM214M.PL15
PVO.4
TRO.11
RHPA4259.7
THRO4156.18
1054_07_TC4_1499
6244_13_B5_4576
62357_14_D3_4589
Median
Mean
STD
Clade*
B (T/F)
C
CRF01_AE
ACD
CD
CD
CD
G
D
D (T/F)
C (T/F)
C (T/F)
BC
C (T/F)
C
B
B
B
B
B (T/F)
B (T/F)
B (T/F)
IC80 Titer in TZM-bl cells, µg/ml
PG9*
PG9/aplaviroc
>50
19.772
>50
18.415
>50
14.409
>50
7.406
>50
8.097
>50
11.544
>50
24.897
>50
30.361
>50
1.571
>50
0.976
>50
3.500
>50
29.579
>50
41.902
>50
4.069
>50
18.933
>50
8.239
>50
35.584
>50
7.092
>50
20.071
>50
36.094
>50
28.640
>50
17.254
17.834
17.655
12.160
IC80 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9*
>100
>333
31.308
>333
33.074
>333
7.363
>333
10.890
>333
19.843
>333
50.284
>333
55.550
>333
2.193
>333
1.298
>333
8.549
>333
20.305
>333
39.067
>333
4.462
>333
40.579
>333
11.173
>333
100.000
>333
19.358
>333
61.850
>333
57.473
>333
46.026
>333
16.826
>333
20.305
30.356
25.118
PG9/aplaviroc
131.815
122.767
96.062
49.373
53.983
76.958
165.983
202.403
10.476
6.510
23.333
197.193
279.345
27.126
126.218
54.930
237.227
47.277
133.804
240.628
190.935
115.025
118.896
117.699
81.066
Figure 7. Graphical representation of the IC80s of aplaviroc and PG9-aplaviroc in the panel of
pseudoviruses poorly neutralized by PG9 (IC80>50 µg/ml).
Statistical analysis for all samples where PG9 has IC80> 50ug/ml.
The following Mean and Median IC80s were calculated by GraphPad Prism
analysis:
PG9/aplaviroc Median IC80 = 118.9nM, Mean IC80 = 117.7nM
Aplaviroc Median IC80 =20.31nM, Mean IC80 =30.36nM.
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC80 values of Aplaviroc and
PG9/aplaviroc (P value 0.0001).
29 Pseudoviruses neutralized by both PG9 and PG9-aplaviroc with IC80>50 µg/ml
(>333nM):
Virus ID
3103.v3.c10
T251-18
Ce2010_F5
QH0692.42
Clade*
ACD
CRF02_AG
C (T/F)
B
IC80 Titer in TZM-bl cells, µg/ml
PG9*
PG9/aplaviroc
>50
>50
>50
>50
>50
>50
>50
>50
IC80 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9*
>100
>333
94.866
>333
>100
>333
98.769
>333
PG9/aplaviroc
>333
>333
>333
>333
Pseudoviruses where PG9-aplaviroc shows decreased potency (IC80) relative to
wildtype PG9:
Decrease of potency of PG9/apl relative to PG9, IC80s
Virus ID
6535.3
SC422661.8
AC10.0.29
REJO4541.67
TRJO4551.58
WITO4160.33
1006_11_C3_1601
1012_11_TC21_3257
6240_08_TA5_4622
SC05_8C11_2344
Du156.12
ZM53M.PB12
CAP210.2.00.E8
HIV-0013095-2.11
HIV-16055-2.3
Ce0393_C3
Ce1176_A3
Ce0682_E4
Ce1172_H1
Ce2060_G9
249M B10
ZM247v1(Rev-)
1394C9G1(Rev-)
Ce704809221_1B3
CNE19
CNE20
CNE21
CNE17
CNE52
CNE53
CNE58
MS208.A1
Q259.d2.17
Q842.d12
0330.v4.c3
0260.v5.c36
191084 B7-19
9004SS_A3_4
T257-31
928-28
263-8
T278-50
T255-34
211-9
235-47
C1080.c03
R2184.c04
R1166.c01
R3265.c06
C3347.c11
C4118.c09
CNE5
BJOX009000.02.4
BJOX015000.11.5
BJOX010000.06.2
BJOX025000.01.1
X1193_c1
X1254_c3
X2131_C1_B5
P1981_C5_3
Clade*
B
B
B
B
B
B
B (T/F)
B (T/F)
B (T/F)
B (T/F)
C
C
C
C
C
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
C (T/F)
BC
BC
BC
BC
BC
BC
BC
A
A
A
A
A
A (T/F)
A (T/F)
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
G
G
G
G
IC80 Titer in TZM-bl cells, µg/ml
PG9*
PG9/aplaviroc
0.627
1.490
7.054
12.585
0.845
1.810
0.163
0.185
4.061
8.795
0.037
0.049
1.906
2.119
0.605
4.402
6.333
10.408
4.283
6.784
0.190
0.342
0.221
0.509
0.173
3.241
0.030
0.170
0.06
0.110
0.055
0.084
0.044
0.117
0.698
1.507
0.206
0.530
0.172
0.297
0.290
0.508
0.323
0.565
0.114
0.155
0.137
1.233
0.146
0.245
0.278
0.637
0.094
0.392
0.484
1.171
0.081
0.821
0.60
3.285
0.122
0.732
0.014
0.409
1.379
1.247
0.081
0.215
0.076
0.159
9.0
10.948
0.202
0.346
0.251
0.484
0.124
0.353
0.450
1.587
1.050
1.784
3.192
5.504
0.134
0.364
0.243
0.834
1.201
1.882
0.013
0.024
1.237
3.136
2.876
4.213
0.535
1.408
0.113
1.065
0.200
0.262
0.051
0.061
8.211
11.028
1.489
5.628
0.740
2.804
0.335
2.721
0.304
1.745
0.304
1.557
0.286
2.383
2.512
5.532
IC80 Titer in TZM-bl cells, nM
Aplaviroc (nM)
PG9*
12.517
4.18
79.320
47.027
87.686
5.633
82.676
1.087
>100
27.073
78.852
0.247
32.098
12.707
98.066
4.034
67.658
42.22
32.815
28.553
93.800
1.267
6.009
1.473
78.371
1.153
7.069
0.2
8.758
0.4
2.152
0.367
87.151
0.293
29.311
4.653
74.695
1.373
17.488
1.147
2.674
1.933
8.042
2.153
12.912
0.760
69.147
0.913
17.066
0.973
8.431
1.853
5.283
0.627
29.677
3.227
>100
0.540
60.248
3.967
51.858
0.813
1.099
0.093
48.616
9.193
>100
0.540
8.747
0.507
97.906
59.953
26.672
1.347
24.314
1.673
68.083
0.827
59.885
3.000
44.140
7.000
43.820
21.280
27.550
0.893
94.925
1.620
8.140
8.007
82.338
0.087
21.270
8.247
27.921
19.173
22.063
3.567
13.964
0.753
3.051
1.333
3.470
0.340
35.617
54.740
45.477
9.927
19.761
4.933
18.749
2.233
16.344
2.027
17.921
2.027
19.084
1.907
71.099
16.747
PG9/aplaviroc
9.933
83.899
12.067
1.233
58.633
0.325
14.129
29.345
69.384
45.227
2.280
3.395
21.608
1.132
0.734
0.561
0.779
10.045
3.535
1.983
3.384
3.765
1.036
8.218
1.630
4.246
2.611
7.810
5.473
21.899
4.880
2.728
8.314
1.434
1.059
72.986
2.308
3.227
2.353
10.577
11.894
36.695
2.426
5.563
12.543
0.162
20.908
28.090
9.384
7.098
1.748
0.409
73.518
37.518
18.695
18.140
11.630
10.381
15.888
36.880
30 X1632_S2_B10
231966.c02
3301.v1.c24
6540.v4.c1
6545.v4.c1
Median
Mean
STD
G
D
AC
AC
AC
0.934
0.257
0.756
0.253
0.351
0.286
1.070
1.943
5.046
0.708
2.568
0.694
1.082
1.082
2.232
2.967
96.596
14.785
24.002
55.967
25.819
27.736
39.210
30.891
6.227
1.713
5.040
1.687
2.340
1.907
7.136
12.953
33.641
4.723
17.120
4.627
7.210
7.210
14.878
19.777
Average decrease of potency of PG9/apl relative to PG9: 2.08 fold
Average improvement of potency for PG9/apl relative to aplaviroc: 2.64 fold
Figure 8. Graphical representation of IC80s in the pseudovirus panel where PG9-aplaviroc has decreased
potency relative to PG9.
Statistical analysis for all samples with decreased PG9/aplaviroc potency
relative to PG9.
The following Mean and Median IC80s were calculated by GraphPad Prism
analysis:
PG9 Median IC80 = 1.91nM, Mean IC80 = 7.14nM
PG9/aplaviroc Median IC80 = 7.21nM, Mean IC80 = 14.88nM
Aplaviroc Median IC80 =27.74nM, Mean IC80 =39.21nM.
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC80 values of PG9 and PG9/aplaviroc (P
value 0.0001).
Mann Whitney test has shown that in this set of samples there is statistically
significant difference between Median IC80 values of Aplaviroc and
PG9/aplaviroc (P value <0.0001).
31 6. PG9 and PG9-aplaviroc MPI full summary.
Special Note: MPIs >99.5% were rounded up by the software analysis program
to 100%.
32 MPI
Virus ID
6535.3
QH0692.42
SC422661.8
PVO.4
TRO.11
AC10.0.29
RHPA4259.7
THRO4156.18
REJO4541.67
TRJO4551.58
WITO4160.33
CAAN5342.A2
Average
WEAU_d15_410_5017
1006_11_C3_1601
1054_07_TC4_1499
1056_10_TA11_1826
1012_11_TC21_3257
6240_08_TA5_4622
6244_13_B5_4576
62357_14_D3_4589
SC05_8C11_2344
Average
Du156.12
Du172.17
Du422.1
ZM197M.PB7
ZM214M.PL15
ZM233M.PB6
ZM249M.PL1
ZM53M.PB12
ZM109F.PB4
ZM135M.PL10a
CAP45.2.00.G3
CAP210.2.00.E8
HIV-001428-2.42
HIV-0013095-2.11
HIV-16055-2.3
HIV-16845-2.22
Average
Ce1086_B2
Ce0393_C3
Ce1176_A3
Ce2010_F5
Ce0682_E4
Ce1172_H1
Ce2060_G9
Ce703010054_2A2
BF1266.431a
246F C1G
249M B10
ZM247v1(Rev-)
7030102001E5(Rev-)
1394C9G1(Rev-)
Ce704809221_1B3
Average
CNE19
CNE20
CNE21
CNE17
CNE30
CNE52
CNE53
CNE58
Average
MS208.A1
Q23.17
MPI summary continued:
Clade*
B
B
B
B
B
B
B
B
B
B
B
B
B(T/F)
B(T/F)
B(T/F)
B(T/F)
B(T/F)
B(T/F)
B(T/F)
B(T/F)
B(T/F)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
C(T/F)
BC
BC
BC
BC
BC
BC
BC
BC
A
A
PG9
96
20
86
78
55
90
63
61
96
95
100
87
77
64
95
0
83
99
97
27
13
93
63
100
99
87
97
0
100
95
100
89
69
100
98
100
100
100
79
88
0
100
100
23
100
100
100
100
100
10
97
100
5
100
100
76
99
100
99
100
16
100
100
100
89
98
100
PG9/Aplaviroc
100
78
96
100
83
99
100
93
99
96
100
97
95
84
100
83
92
99
98
89
97
100
94
100
100
98
100
94
100
100
100
100
93
100
100
100
100
100
96
99
100
100
100
68
100
100
100
100
100
100
100
100
92
100
100
97
100
100
100
100
81
100
99
100
98
100
100
33 Q461.e2
Q769.d22
Q259.d2.17
Q842.d12
0330.v4.c3
0260.v5.c36
Average
191955_A11
191084 B7-19
9004SS_A3_4
Average
T257-31
928-28
263-8
T250-4
T251-18
T278-50
T255-34
211-9
235-47
Average
620345.c01
C1080.c03
R2184.c04
R1166.c01
R3265.c06
C2101.c01
C3347.c11
C4118.c09
CNE5
BJOX009000.02.4
Average
BJOX015000.11.5
BJOX010000.06.2
BJOX025000.01.1
BJOX028000.10.3
Average
X1193_c1
P0402_c2_11
X1254_c3
X2088_c9
X2131_C1_B5
P1981_C5_3
X1632_S2_B10
3016.v5.c45
A07412M1.vrc12
231965.c01
231966.c02
191821_E6_1
Average
3817.v2.c59
6480.v4.c25
6952.v1.c20
6811.v7.c18
89-F1_2_25
Average
3301.v1.c24
6041.v3.c23
6540.v4.c1
6545.v4.c1
Average
0815.v3.c3
3103.v3.c10
Average
A
A
A
A
A
A
A(T/F)
A(T/F)
A(T/F)
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF02_AG
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
CRF01_AE (T/F)
G
G
G
G
G
G
G
D
D
D
D
D (T/F)
CD
CD
CD
CD
CD
AC
AC
AC
AC
ACD
ACD
91
99
90
100
100
94
97
100
100
100
100
100
100
99
100
42
97
97
99
97
92
67
100
98
98
100
99
100
100
100
96
96
98
97
93
84
93
100
95
98
16
99
95
94
85
66
90
80
100
100
100
99
100
100
95
99
100
100
100
100
100
100
100
100
70
99
100
100
100
97
96
100
100
98
100
100
100
100
100
97
99
100
99
100
99
100
100
100
100
91
100
97
95
98
100
99
94
100
78
83
97
13
35
3
88
47
100
94
99
98
98
0
58
29
100
99
100
97
96
90
100
97
100
100
99
100
100
98
69
84
34