anti-CMYC (ICL inc). Typically, libraries were
sorted three to five times, and the enriched
cell populations were frozen until sequencing
could be carried out as described previously
( 22 ). Mutations found in the most enriched
clones were incorporated into the most recent
designs, synthesized at Genscript either as
C-terminal His tagged gp120s or MD39/MD64
trimers in the pHLsec vector and expressed
and purified as described previously ( 22 ).
Nanoparticle design and purification
To obtain multivalent immunogens, trimers were
genetically fused to ferritin fromHelicobacter
pyloriusing a short flexible linker. Genes were
codon optimized for HEK293 cells and cloned
into the pHLsec plasmid (GenScript). MD39-
NP DNA was cotransfected with a plasmid en-
coding human Furin protease into FreeStyle
293F cells (Invitrogen, Cat no. R79007) using
293Fectin (ThermoFisher) and proteins were
expressed at 37°C for 4 days. NPs were purified
either using snow drop lectin-conjugated agar-
ose beads (Vector laboratories) or HiTrap NHS-
Activated HP affinity columns (GE Healthcare)
conjugated with PGT145, each followed by
gel-filtration using a Superose 6 size-exclusion
chromatography column (GE Healthcare). N332-
GT2 NP had the Furin cleavage site replaced
by a flexible linker (SHSGSGGSGSGGHA) as
well as an L545P mutation, both discovered by
library screening; hence, N332-GT2 NP was
not cotransfected with Furin. NP-assembly
was assessed by negative-stain EM and SEC +
multiangle light scattering (SEC-MALS) using
a Superose 6 10/300 column (GE Healthcare) at
a flow rate of 0.5 ml/min followed by DAWN
HELEOS II and Optilab T-rEX detectors (Wyatt
Technology), correcting for the glycan molec-
ular mass by applying thebuilt-inprotein-
conjugate analysis (ASTRA).
Neutralization activity
Neutralizing activity of monoclonal antibodies
(mAbs) was assessed using a single round of
replication in TZM-bl target cells, in the ab-
sence of DEAE-dextran except for the assays
in Fig. 2K, as described previously ( 34 ). Briefly,
pseudoviruses were generated by cotransfec-
tion of HEK293T cells with an Env-expressing
plasmid and an Env-deficient genomic back-
bone plasmid (pSG3DEnv).
Cryo-EM structure determination
High resolution cryo-EM structures were deter-
mined for four complexes: (i) MD39 + BG18;
(ii) N332-GT2 + BG18iGL; (iii) N332-GT5 + HMP1;
and (iv) N332-GT5 + HMP42. Of note, initial
attempts to determine even low-resolution EM
structures of HMP Fabs bound to N332-GT2
were not successful. However, in the late stages
of this study, we found that the N332-GT5 tri-
mer proved capable of forming stable com-
plexes with the two clones, HMP1 and HMP42,
representing BG18-type I and type II precur-
sors, respectively. In general, trimers were
incubated with a 6-10× molar excess of Fab
overnight at room temperature. Complexes
containing HMP1 or HMP42 also included
RM20A3 Fab, a non-neutralizing trimer base-
binding antibody that helps increase orienta-
tion sampling of the particles. The following
morning, each complex was purified using a
HiLoad 16/600 Superdex 200pg size-exclusion
column (GE Healthcare) with Tris-buffered
saline (50 mM Tris pH 7.4, 150 mM NaCl) as
the running buffer, and the peak correspond-
ing to trimer-Fab complex was pooled and
concentrated to ~6 to 8 mg/ml. 3.5ml of each
complex was mixed with 0.5ml of 0.42 mM
n-dodecylb-D-maltoside (DDM; Anatrace),
such that final DDM concentration (0.06 mM)
is below the critical micellar concentration
(CMC). A 4-ml aliquot of the complex was
applied to a C-Flat grid (CF-2/2-4C, Electron
Microscopy Sciences, Protochips, Inc.) or
Quantifoil grid (Q 1.2/1.3-4C, Quantifoil Micro
Tools GmBH), which had been plasma cleaned
for 10 s using a mixture of Ar/O 2 (Gatan Solarus
950 Plasma system), and following a 10-s in-
cubation, the grid was blotted between 4 to
6 s and plunged into liquid ethane using an
FEI Vitrobot Mark IV (100% relative humid-
ity, 10°C).
The samples were imaged using either a
Thermo Fisher Titan Krios operating at 300 kV
or a Thermo Fisher Talos Arctica operating at
200 kV, both with a Gatan K2 Summit direct
electron director operating in counting mode.
Automateddatacollectionwasperformedusing
the Leginon software suite ( 35 ). Each micro-
graph movie (250-ms exposure per frame) was
collected at a magnification of 29,000× and a
pixel size of 1.03 Å (Krios) or 36,000× and a
pixel size of 1.15 Å (Arctica). Data collection
statistics for each sample are summarized in
table S1. Micrograph movie frames were aligned
and dose-weighted using MotionCor2 ( 36 ), and
CTF models were calculated using GCTF ( 37 ).
Single particles were selected using DoGPicker
( 38 ) from the whole-frame aligned and summed
micrographs, and particles extracted using
Relion 3.0 ( 39 )usingaboxsizeof288or
320 pixels. After numerous rounds of 2D and
3D classification, final reconstructions were
performed in Relion 3.0, and after postpro-
cessing, the final resolution estimates (FSC
0.143) are ~3.9 Å for N332-GT2 + BG18iGL
(C3 symmetry),~4.4 Å for MD39 + BG18 (C3
symmetry), ~3.7 Å for N332-GT5 + HMP1 +
RM20A3 (C3 symmetry), and ~3.4 Å for N332-
GT5 + HMP42 + RM20A3 (asymmetric). Addi-
tional data processing statistics are summar-
ized in fig. S3.
Atomic models were built and refined into
the high-resolution reconstructions by creat-
ing homology models based off deposited
coordinates of BG505 SOSIP.664 (PDB 5cez)and 354BG18 Fab (PDB 5ud9), as well as dock-
ing of an HMP42 Fab crystal structure from
this study (table S6), followed by an iterative
cycle of manual building in COOT ( 40 ) and
real space refinement in Phenix 1.13 ( 41 ) and
Rosetta Relax 3.10 ( 42 ). Glycans were validated
by CARP ( 43 ) and Privateer ( 44 ), and overall
structures were evaluated using EMRinger
( 45 )andMolProbity( 46 ). Buried surface area
calculations were performed using UCSF
Chimera ( 47 ).ELISA
For analysis of serum responses from
immunized mice
N332-GT2–specific antibody titers were de-
tected by ELISA, using anti-His Ab (2mg/ml)
to capture N332-GT2 or N332-GT2-KO antigen
(2mg/ml) on the plate. Mouse sera were in-
cubatedfor2hoursandalkalinephospha-
tase conjugated anti-mouse IgG (Jackson
ImmunoResearch, #115-055-071) was incu-
bated another hour. Titers were determined
from the dilution curve in the linear range of
absorbance. All noncommercial ELISA plates
were developed with p-Nitrophenyl Phosphate
(Sigma, # N2770). Absorbance at 405 nm was
determined with a plate reader (BioTek).For analysis of mAb binding
mAb binding ELISAs were performed by cap-
turing antigen (1 μg/ml) onto plates precoated
with anti-His antibody (1 μg/ml; Genscript)
and blocked with blocking buffer (5% skim
milk, 1% fetal bovine serum, 0.2% tween 20 in
PBS). Dilution series of mAbs were added
as indicated and labeled with peroxidase-
conjugated goat anti-mouse IgG (1:5000;
Jackson ImmunoResearch). Wells were devel-
oped with 1-Step Ultra TMB-ELISA substrate
(Thermo Scientific) diluted 1:4 in H 2 Oand
stopped by addition of 0.5M H 2 SO 4. Absorb-
ance was read at 450 nm and reference ab-
sorbance measured at 570 nm was subtracted
from each well.Surface plasmon resonance (SPR)
Kinetics and affinities of antibody-antigen in-
teractions were measured on a ProteOn XPR36
(Bio-Rad) using GLC Sensor Chip (Bio-Rad)
or Biacore4000 (GE) with Series S Sensor
Chip CM5 (GE). We used 1× HBS-EP+ pH 7.4
running buffer (20× stock from Teknova,
Cat. No. H8022) supplemented with BSA at
1 mg/ml. Following manufacturer’sinstruc-
tions for Human Antibody Capture Kit (Cat.
No. BR-1008-39 from GE), we immobilized
about 6000 RUs of capture mAb onto each
flow cell of GLC Sensor Chip or about 10,000
RUs in the case of the CM5 Sensor Chip. In a
typical experiment on the ProteOn XPR36
system, about 300 to 400 RUs of mAbs were
captured onto each flow cell, and analytes
were passed over the flow cell at 50ml/min forSteichenet al.,Science 366 , eaax4380 (2019) 6 December 2019 10 of 13
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