formic acid). A 5-ml aliquot of the sample
(~1.5mM) was injected onto C18 PepMap
300 column (300mm i.d. × 15 cm, 300 Å,
Thermo Fisher Scientific) at a flow rate of
4 ml/min. The following CH 3 CN/H 2 O multi-
step gradient was used: 3% B for 5 min,
followed a linear increase to 40% B in 50 min,
then a linear increase to 90% B in 15 min. The
column was held at 97% B for 10 min before
reequilibration. All mass spectrometric analy-
sis was performed in the positive ion mode
using data-dependent acquisition mode: The
five most intense ions in the survey scan in
the mass range 400 to 2000m/zwere se-
lected for alternating CID and ETD in the
LTQ linear ion trap using a normalized col-
lision energy of 30% for CID and an ion-ion
reaction time of 100 to 150 ms for ETD. Full
MS scans were measured at a resolution (R)
of 30,000 atm/z400. Under these condi-
tions, the measured R (FWHM) in the orbi-
trap mass analyzer is 20,000 atm/z 1000
and 17,000 atm/z1500.
Glycopeptide identification and disulfide
bond analysis
Details of the glycopeptide compositional anal-
ysis have been described ( 63 – 65 ). Briefly, com-
positional analysis of glycopeptides was carried
out by first identifying the peptide portion
from tandem MS data. Once the peptide por-
tion was determined, plausible glycopeptide
compositions were obtained using the high-
resolution MS data and GlycoPep DB. The
putative glycopeptide composition was con-
firmed manually from CID and ETD data.
Disulfide bond patterns of CH848 10.17DT
SOSIP Envs were determined by mapping the
disulfide-linked peptides. Data analysis was
performed using Mascot (v 2.5.1) search engine
for peptides containing free cysteine residues
and disulfide bond patterns were analyzed
manually as described ( 66 , 67 ).
In vitro HIV-1 neutralization
Antibody-mediated HIV-1 neutralization was
measured using Tat-regulated luciferase (Luc)
reporter gene expression to quantify reduc-
tions in virus replication in TZM-bl cells as
described ( 68 ). TZM-bl cells were obtained
from the NIH AIDS Research and Reference
Reagent Program, as contributed by J. Kappes
and X. Wu. The monoclonal antibody or serum
was pre-incubated with virus (~150,000 rela-
tivelightunitequivalents)for1hourat37°C,
and TZM-bl cells were subsequently added.
After 48 hours, cells were lysed and Luc activ-
ity determined using a microtiter plate lumi-
nometer and BriteLite Plus Reagent (Perkin
Elmer). Neutralization titers are the antibody
concentration inmg/ml or serum reciprocal
dilution at which relative luminescence units
(RLU) were reduced by 50% compared to RLU
in virus control wells after subtraction of back-
ground RLU in cell control wells (IC 50 and
ID 50 , respectively).Surface plasmon resonance (SPR)
SPR experiments were performed on a BIACore
T200. To determine apparent affinities, ap-
proximately 300 RU (range 310 to 321 RU)
of each antibody was captured on an anti-
human IgFc immobilized Series S CM5 sensor
chip (GE Healthcare). Serial dilutions of SOSIP
Env was flowed over immobilized antibody
in HEPES-buffered saline. To determine bind-
ing affinity, biotinylated SOSIP gp140 was im-
mobilized on streptavidin-coated sensor chips.
Serial dilutions of antibody Fab were flowed
over the Env. Each concentration of Env was
flowed over each immobilized antibody for
120 s and dissociation was measured for 600 s.
In between injections of each Env concentra-
tion, the surface was regenerated by injecting
glycine pH 2 for 30 s. Binding rate constants
(ka,kd) were measured after global curve fitting
to a Langmuir model. Curve fitting analysis
was performed with BiaEvaluation software
(GE Healthcare) using a 1:1 Langmuir model,
or a heterogeneous binding model when ap-
propriatetoderiverate(ka,kd)andapparentor
true equilibrium dissociation constants (Kd).Serum IgG ELISA binding titers
Avi-tag SOSIP capture or PGT151 SOSIP cap-
ture ELISAs were performed as described
( 69 ). Protein (2mgml–^1 ) in sodium bicarbonate
buffer was incubated in sealed Nunc-absorp
(ThermoFisher) plates overnight at 4°C. Un-
boundproteinwaswashedawayandtheplates
were blocked with SuperBlock for 1 hour. Serial
dilution of serum or monoclonal antibodies
were added to the plate for 90 min. Binding
antibodies were detected with HRP-labeled
anti-IgG Fc antibody. HRP was detected with
3,3′,5,5′-tetramethylbenzidine. Binding titers
were analyzed as area-under-curve of the log-
transformed concentrations.Serum IgG and monoclonal antibody
competition ELISAs
Serum IgG or monoclonal antibody compe-
tition assays were performed as described
( 61 , 70 ). In brief, Nuncsorp plates were coated
with HIV-1 envelope, washed and blocked as
stated above for direct ELISAs. After block-
ing was complete, mouse serum was diluted in
SuperBlock at a 1:50 dilution and incubated
in triplicate wells for 90 min. For monoclonal
antibody competitors, a dilution series start-
ingat100mgml–^1 was incubated in triplicate
wells for 90 min. Non-biotinylated monoclo-
nal antibodies were incubated with the Env
in triplicate as positive controls for blocking.
To determine relative binding, no plasma or
no antibody was added to a group of wells
scattered throughout the plate. After 90 min,
the non-biotinylated antibody or plasma waswashed away, and biotinylated monoclonal
antibodies was incubated in the wells for
1 hour at sub-saturating concentrations. Each
well was washed, and binding of biotinylated
monoclonal antibodies was determined with
a 1:8000 dilution of horseradish peroxidase
(HRP)–conjugated streptavidin. HRP was de-
tected with tetramethylbenzidine and stopped
with 1% HCl. The absorbance at 450 nm of
each well was read with a Spectramax plate
reader (Molecular Devices). Binding of the
biotinylated monoclonal antibody to HIV-1
envelope in the absence of plasma was com-
pared to in the presence of plasma to calculate
percent inhibition of binding. Based on his-
torical negative controls, assays were consid-
ered valid if the positive control antibodies
blocked greater than 20% of the biotinylated
antibody binding.Sortase A conjugation of HIV-1 envelope trimers
to ferritin nanoparticles
CH848 10.17DT SOSIP gp140 was expressed
with amino acids LPSTGG encoded at its C
terminus. The CH848 10.17DT SOSIP was ex-
pressed in Freestyle293 cells and purified by
affinity chromatography with broadly neu-
tralizing trimer-specific antibody PGT151 as
stated above. Trimeric gp140 was isolated by
size-exclusion chromatography using a Superose6
16_60 column. Ferritin particles were expressed
with a (GGGGG)3 repeat sequence encoded
at the N terminus of each subunit. For affinity
purification of ferritin particles, 6×His tags
were appended to the C terminus. CH848 SOSIP
with a C-terminal sortase A tag and ferritin
particles with a sortase A N-terminal tag were
buffer-exchanged into 50 mM Tris, 150 mM
NaCl, 5 mM CaCl 2 ,pH7.5.120mMSOSIPgp140
was mixed with 120mM ferritin subunits and
incubated with 100mM sortase A overnight at
room temperature. After incubation, conju-
gated particles were isolated from free ferritin
or free SOSIP gp140 by size-exclusion chro-
matography using a Superose6 16_60 column.Antibody binding to HIV-1 envelope by biolayer
interferometry (BLI)
Biolayer interferometry was performed as
described ( 71 ). Antibodies were immobilized
to anti-Fc sensor tips and incubated with
50 mgml–^1 of Env SOSIP gp140-nanoparticle
or SOSIP trimer solutions for 400 s. To com-
pare binding profiles between Env SOSIP
gp140 and Env SOSIP gp140 nanoparticles,
BLI binding values were normalized to PGT151
binding response by dividing each antibody
binding response value by the PGT151 binding
response value.Antigen-specific fluorescence-activated
single B cell sorting
To sort Env-specific memory B cells, spleno-
cytes were incubated with the followingSaunderset al.,Science 366 , eaay7199 (2019) 6 December 2019 13 of 17
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